Human Hematopoietic Cell Lines Research Articles

Effects of notoginosides on proliferation and upregulation of GR nuclear transcription factor in hematopoietic cells

To investigate the effects of panax notoginosides (PNS) on the proliferation of human hematopoietic stem/progenitor cells, and to explore the signaling pathway of the nuclear transcription factor of the glucocorticoid receptor (GR-NTF) initiated by PNS related with the proliferation. The human CD34+ cells and bone marrow nuclear cells were exposed to PNS at a concentration of 0, 10, 25, 50, and 100 mg/L, respectively, in semi-solid culture system to observe colony forming unite of all lineages, granulocyte, erythrocyte, and megakaryocyte (CFUGEMM, CFU-GM, CFU-E, and CFU-MK). Three lineages of human hematopoietic cell lines, including granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF- 288, and Meg-01 cells were incubated with PNS at 20 mg/L for 14 d. Meanwhile, dexamethasone (Dex) was used as a positive control. The nuclear protein of the cells was analyzed by Western blotting with monoclonal antibodies against the amino or carboxyl terminus of GR-NTF. Electrophoretic mobility shift assay performed by using the 32P-radiolabeled GR-NTF consensus oligonucleotide. PNS promoted the proliferation of CD34+ cells and significantly raised the colony numbers of CFU-GEMM by 34.7%+/-16.0% over the non-PNS control (P<0.01). PNS also enhanced the proliferation of CFU-GM, CFU-E, and CFU-MK by 39.3%+/- 5.7%, 33.3%+/-7.3%, and 26.2%+/-3.2%, respectively. GR-NTF protein levels of either the amino or carboxyl terminus in K562, CHRF-288, and Meg-01 treated by PNS increased by 2.4-2.8 fold and 1.3- 3.9 fold over the untreated cells. GR-NTF binding activity, initiated by either PNS or Dex, was apparently elevated to form the complex of GR-NTF with DNA as higher density bands in K562 and CHRF-288 cells, and some activity appeared as a band in HL-60 cells induced by PNS. PNS displayed the action of hematopoietic growth factor-like or synergistic efficacy to promote proliferation of human progenitor cells, may play a role in the upregulation of gene expression related to proliferation of hematopoietic cells through increasing the GR-NTF synthesis and its DNA binding activity.

HCELL Is the Major E- and L-Selectin Ligand Expressed on Human Hematopoietic Progenitor Cells and Colon Carcinoma Cells.

Selectins are a class of cell surface proteins specialized to mediate adhesive interactions under hydrodynamic shear flow conditions, and are critical in the trafficking of hematopoietic progenitor cells (HPC) into the bone marrow and dissemination of cancer cells in hematogenous metastasis, among other biological events. More specifically, E-selectin expressed by endothelial cells has been shown to mediate shear-resistant tethering/rolling of cells recruited from the fluid stream, while L-selectin expressed by distinct leukocyte subsets has been variously shown to mediate lymph node homing, as well as the formation of heterotypic cell aggregates in bulk flow. We have previously reported that the primitive CD34+ human hematopoietic progenitor cell (HPC) line KG1a exhibits stronger E-/L-selectin ligand activities than HL60, a more differentiated CD34− human hematopoietic cell line. These findings were attributed to the HPC/KG1a restricted expression of the sialofucosylated CD44 glycoform termed HCELL (Hematopoietic Cell E-/L-selectin Ligand), since both cell lines express approximately equivalent levels of CD44 and the well-characterized selectin ligand P-selectin glycoprotein ligand-1 (PSGL-1). To directly assess whether HCELL functions as the high affinity E-/L-selectin ligand on KG1a cells, HCELL expression was silenced using lentiviral siRNA targeting CD44, and the E-/L-selectin ligand activities were subsequently compared between untreated and transduced KG1a cells. CD44 siRNA transduction of KG1a cells decreased CD44 (i.e. HCELL) expression levels >90% mean fluorescence intensity (MFI) relative to untreated and vector alone-transduced cells as measured by flow cytometry. CD44 targeting was specific, as expression of PSGL-1 was not reduced upon CD44 siRNA transduction, nor were CD29, CD49d, or CD49e levels affected. Functionally, at venous shear stress levels (≤4 dyn/cm2), CD44 siRNA-transduced KG1a cells rolled markedly faster than untreated and vector alone-transduced cells on a monolayer of E-selectin transfected Chinese hamster ovary (CHO-E) cells. At arterial and pathological shear (>4 dyn/cm2), control cells maintained rolling interactions on CHO-E cells, whereas CD44 siRNA-transduced cells readily detached from the monolayer. Comparable results were obtained for L-selectin-dependent peripheral blood mononuclear cell rolling on CD44 siRNA-transduced versus untreated and vector alone-transduced KG1a cells. Furthermore, the HCELL-negative HL60 cells supported similarly weak binding of E- and L-selectin as CD44 siRNA-transduced KG1a cells. Collectively, the data directly show that HCELL is the predominant ligand that stabilizes shear-resistant HPC binding to E- and L-selectin. We have also previously reported that the human colon carcinoma cell line LS174T expresses HCELL. To examine whether HCELL on LS174T cells functions as a selectin ligand, we again suppressed expression through targeted gene silencing by lentiviral CD44 siRNA transduction. Similar to KG1a cells, specific CD44/HCELL silencing on LS174T cells was achieved (>90% reduction in MFI relative to untreated and vector controls), resulting in significant inhibition of E- and L-selectin binding capabilities at physiologically relevant shear stress levels. In summary, these findings demonstrate that HCELL is the most potent E-/L-selectin ligand mediating shear-resistant adhesive interactions on human cells.

Essential Role for Activated Leukocyte Cell Adhesion Molecule Gene Silencing by GATA-1 in Megakaryocytic Progenitor Cell Biology.

Regulation of hematopoietic progenitor cell lineage-commitment, proliferation and differentiation by cell-cell adhesion mechanisms is poorly understood. Activated leukocyte cell adhesion molecule (ALCAM) is a member of the immunoglobulin super family. It is expressed by human hematopoietic stem cells, bone marrow stromal cells, endothelial cells and osteoblasts. Monoclonal anti-ALCAM antibodies inhibit myeloid but not erythroid colony formation, which suggest a lineage-specific role for ALCAM in hematopoiesis. To explore this hypothesis, ALCAM mRNA and protein expression was quantified in human hematopoietic cell lines of myeloid, lymphoid, erythroid, and megakaryocytic lineages by real-time quantitative PCR and western blot analyses. No ALCAM transcripts were detected in K562 and MEG-01 cells, the level of ALCAM mRNA was 2-fold more abundant in HL-60 and THP-1 cells than in U937 and Jurkat cells. This expression pattern was confirmed at the protein level as none of the megakaryocyte-erythroid progenitor cell lines (K562, MEG-01 and HEL) expressed ALCAM. On the contrary, ALCAM was abundantly expressed in THP-1 and HL-60 cells and moderately in U937 and Jurkat cells. GATA-1 was abundantly expressed in megakaryocyte-erythroid progenitor cell lines but not in any of the myeloid cell lines. Thus, there is an inverse relationship between expression of ALCAM and GATA-1 in hematopoietic cells. To test the hypothesis that GATA-1 is involved in silencing ALCAM gene expression, multiple ALCAM-promoter luciferase constructs were studied. A negative regulatory region was identified in the ALCAM promoter containing an inverted GATA-1 cis element at −850 upstream of the translational start site. GATA-1 occupied this canonical element in vivo as determined by chromatin immunoprecipitation experiments. A two-base pair mutation of the −850 GATA-1 cis element increased ALCAM promoter activity 3-fold in K562 and MEG-01 cells, providing direct evidence of GATA-1's negative regulatory role in ALCAM promoter activity. To test the hypothesis that ALCAM silencing is essential for megakaryocyte-erythroid progenitor cell biology, stable lines of K562 cells were established forcibly expressing ALCAM-GFP or a control GFP. Live cell imaging demonstrated recruitment of ALCAM to sites of cell-cell adhesion in ALCAM-GFP-K562 cells, whereas GFP remained distributed in the cell cytosol in control cells. ALCAM-GFP-K562 cells formed markedly more clusters consisting of significantly more cells than control GFP-K562 cells. Finally, the number of ALCAM-GFP-K562 cells at log-phase growth was significantly higher than GFP-K562 cells over the same time period. Our findings demonstrate for the first time lineage-specific silencing of the cell adhesion molecule ALCAM in megakaryocyte-erythroid progenitor cells, mediated at least in part by GATA-1. That ectopic expression of ALCAM increased proliferation of K562 cells suggests that GATA-1-mediated silencing of ALCAM is essential in slowing down expansion of megakaryocyte-erythroid progenitor cells. Indeed, preliminary studies show an excessive number of erythroid and megakaryocytic cells in the adult spleen of ALCAM-null mice. This model is being used in ongoing studies to confirm our findings in vivo.

A Helper-Dependent Capsid-Modified Adenovirus Vector Expressing Adeno-Associated Virus Rep78 Mediates Site-Specific Integration of a 27-Kilobase Transgene Cassette

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCR-rep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular "nicking" enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.

Stable gene transfer to human CD34 + hematopoietic cells using the Sleeping Beauty transposon

Methods of gene transfer to hematopoietic stem cells that result in stable integration may provide treatments for many inherited and acquired blood diseases. It has been demonstrated previously that a gene delivery system based on the Sleeping Beauty (SB) transposon can be derived where a plasmid transiently expressing the SB transposase can mediate the stable chromosomal integration of a codelivered second plasmid containing a gene expression unit flanked by the inverted repeats derived from the transposon. Plasmid DNA containing the elements required for SB transposition was delivered to hematopoietic cells via electroporation. Integrated transgene (enhanced green fluorescent protein [eGFP]) expression was assessed in vitro and in vivo. In the K562 human hematopoietic cell line, we observed stable expression of eGFP in >60% of cells for over 2 months after electroporation of the two plasmids; in contrast, in control cells either not treated with transposase or exposed to a defective mutant transposase, the level of gene expression had fallen to near background (<0.1%) by 2 weeks. In purified human cord blood CD34(+) progenitor cells, the transposase led to stable gene transfer at levels up to 6% for over 4 weeks, but gene transfer to more primitive nonobese diabetic/severe combined immunodeficient repopulating cells or CD34(+)/CD38(-) in long-term culture was low and electroporation of the cells with plasmid DNA caused significant cell death. The long-term stable expression highlights the potential of this transposase-based gene delivery method for ameliorating diseases affecting the hematopoietic system, although further improvements in gene transfer efficacy are needed.

The α Subunit of the Granulocyte-Macrophage Colony-stimulating Factor Receptor Interacts with c-Kit and Inhibits c-Kit Signaling

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates hematopoiesis and the function of mature host defense cells through the GM-CSF receptor (GMR), which is composed of alpha (alphaGMR) and beta (betaGMR) subunits. Stem cell factor is another important hematopoietic cytokine that signals through c-Kit, a receptor tyrosine kinase, and regulates hematopoietic stem cell maintenance and erythroid development. Like other cytokine receptors, GMR and c-Kit are generally deemed as independent adaptor molecules capable of transducing cytokine-specific signals. We found that the alphaGMR directly interacts with c-Kit and that the interaction is mediated by the cytoplasmic domains. Furthermore, alphaGMR inhibited c-Kit auto-phosphorylation induced by the ligand stem cell factor. Consistent with the inhibitory effect, the expression of alphaGMR was suppressed in cells whose viability was dependent on c-Kit signaling. In contrast, the alternatively spliced alpha2 isoform of the alphaGMR could not inhibit c-Kit signaling, providing a rationale for the existence of the alpha2 isoform. Our results suggest that in addition to having the commonly appreciated roles in cytokine signal transduction, the receptors alphaGMR and c-Kit could interact to coordinate their signal initiation.

Survivin silencing sensitizes imatinib-resistant CML cells to the cytotoxic effect of hydroxyurea

13119 Background: Imatinib Mesylate (IM), a semi-specific inhibitor of the BCR-ABL tyrosine kinase, is currently the treatment of choice for Chronic Myeloid Leukemia (CML). However, about one third of CML patients treated with IM develop resistance to the drug because of reactivation of BCR-ABL kinase activity. This phenomenon is usually ascribed to the amplification of the BCR-ABL gene or to the selection of leukemic clones harboring point mutations that abrogate IM binding. To identify novel anti-apoptotic signaling pathways employed by BCR-ABL and devise strategies capable of killing IM-resistant CML cells, we investigated the interplay between BCR-ABL and the Inhibitor of Apoptosis Protein Survivin. Methods: Murine hematopoietic cells (32D) transduced with p210 BCR-ABL and human cell lines either positive (K562, KCL22, KYO1 and LAMA84) or negative (HL60) for the BCR-ABL oncoprotein, were analyzed for Survivin expression by western blot before and after IM treatment. Three different pathways (MAPK, PI3K and JAK2/STA3) potentially involved in BCR-ABL-mediated induction of Survivin were studied using inhibitors specific for each signaling cascade. The effect of Survivin on the proliferation and viability of IM-sensitive and IM-resistant CML cells was investigated after silencing Survivin expression with small interfering RNAs. Results: BCR-ABL tyrosine kinase activity induced an over-expression of Survivin in both human and murine hematopoietic cell lines. This over-expression was both at the transcriptional and the translational level and required the JAK2/STAT3 pathway. Survivin silencing by siRNA increased IM cytotoxicity in IM-sensitive cells but failed to restore IM efficacy in IM-resistant cells. However, Survivin silencing sensitized CML cells to the cytotoxic effect of hydroxyurea and enhanced the efficacy of this compound on three different murine cell lines are insensitive to IM because of point mutations in the BCR-ABL kinase domain (Ba/F3p210Y253F, Ba/F3p210D276G and Ba/F3p210T315I). Conclusions: Reduction of Survivin expression improves the efficacy of IM and increases the sensitivity of IM-resistant CML cells to hydroxyurea. Survivin may represent an attractive therapeutic target for both IM-sensitive and IM-resistant CML patients. No significant financial relationships to disclose.

Human telomerase reverse transcriptase protects hematopoietic progenitor TF-1 cells from death and quiescence induced by cytokine withdrawal

Telomerase is a complex ribonucleoprotein enzyme that exhibits elevated activity in the majority of cases of human leukemia. We have previously shown that retroviral expression of the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), in human cord blood CD34+ cells leads to an enhanced survival of mature hematopoietic cells. The mechanism for this pro-survival effect is not known. Here, we show that telomerase may play a role in leukemogenesis as a survival factor, independent of its role in maintaining telomere length. Retroviral expression of hTERT in the cytokine-dependent, human hematopoietic progenitor cell line, TF-1, resulted in the survival of cells following the withdrawal of cytokine, with protection from apoptosis, but did not promote unlimited replicative potential. This hTERT-mediated effect on cell survival does not involve Bcl-2 family members, results in accumulation of cells in G1 and appears to operate via autocrine expression of IL-3 and activation of the p53/p21 pathway. Survival in the absence of cytokine stimulation was also observed following retroviral expression of hTERT in normal cord blood CD34+ cells. This study demonstrates a novel pro-survival role for hTERT and may have important implications for the role of hTERT in the pathogenesis of leukemia and drug resistance.

Intracellular Traffic and Fate of Protein Transduction Domains HIV-1 TAT Peptide and Octaarginine. Implications for Their Utilization as Drug Delivery Vectors

Transduction domains such as those derived from the HIV-TAT protein are candidate vectors for intracellular delivery of therapeutic macromolecules such as DNA and proteins. The mechanism by which they enter cells is controversial, and very little spatial information regarding the downstream fate of these peptides from the plasma membrane is available. We studied endocytic traffic of fluorescent conjugates of HIV-TAT peptide and octaarginine in human hematopoietic cell lines K562 (CD34-) and KG1a (CD34+) and substantiated our findings in epithelia cells. Both peptides were efficiently internalized to endocytic pathways of both hematopoietic cell lines; however, comparative analysis of the intracellular location of the peptides with endocytic probes revealed major differences in spatial organization of their endocytic organelles and their interaction with the peptides at low temperatures. Double labeling confocal microscopy demonstrates that prelabeled lysosomes of all the tested cells are accessible to internalized peptides within 60 min of endocytic uptake. Incubation of cells with nocodazole and cytochalasin D inhibited peptide traffic from early to late endosomal structures, demonstrating a cytoskeletal requirement for lysosomal delivery. Disruption of Golgi and endoplasmic reticulum dynamics was without effect on peptide localization, suggesting that endosomes and lysosomes rather than these organelles are the major acceptor compartments for these molecules.

A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin

FLRG and follistatin belong to the family of follistatin proteins involved in the regulation of various biological effects, such as hematopoiesis, mediated by their binding to activin and BMP, both members of the TGFbeta family. To further characterize the function of FLRG, we searched for other possible functional partners using a yeast two-hybrid screen. We identified human fibronectin as a new partner for both FLRG and follistatin. We also demonstrated that their physical interaction is mediated by type I motifs of fibronectin and follistatin domains. We then analyzed the biological consequences of these protein interactions on the regulation of hematopoiesis. For the first time, we associated a biological effect with the regulation of human hematopoietic cell adhesiveness of both the type I motifs of fibronectin and the follistatin domains of FLRG and follistatin. Indeed, we observed a significant and specific dose-dependent increase of cell adhesion to fibronectin in the presence of FLRG or follistatin, using either a human hematopoietic cell line or primary cells. In particular, we observed a significantly increased adhesion of immature hematopoietic precursors (CFC, LTC-IC). Altogether these results highlight a new mechanism by which FLRG and follistatin regulate human hematopoiesis.

Expression of Akt (protein kinase B) and its isoforms in malignant lymphomas

Akt (protein kinase B) is a serine/threonine kinase involved in the regulation of cell survival signals. Akt is expressed in T- and B-lymphocytes and is activated in response to cytokine and antigen-receptor stimulation. Three isoforms of Akt have been identified, Akt-1, -2 and -3, but the expression pattern and specific functions of each have not yet been determined for many cell types. To determine whether Akt signaling is enhanced in human malignant lymphomas and to analyse the expression pattern of Akt isoforms in these neoplasms, Akt-1, -2 and -3 expression was studied in 38 cell lines derived from hematopoietic neoplasms, by RT-PCR and western blot analysis. The level of phosphorylated (active) Akt was also analysed in cell lines as well as in 72 human malignant non-Hodgkin's lymphoma tissues. The results suggest that there is constitutive activation of Akt in the majority of primary human lymphomas and hematopoietic cell lines and support its proposed key role in lymphoma cell survival.

Unexpected Evidence That Dimethylsulphoxide (DMSO) Upregulates Expression of CXCR4 on Hematopoietic Stem/Progenitor Cells (HSPC), Increases Their Responsiveness to an SDF-1 Gradient and Enhances Homing to Bone Marrow.

Cryopreservation of bone marrow (BM), mobilized peripheral blood (mPB) and cord blood (CB) cells is a routine procedure to store hematopoietic stem/progenitor cells (HSPC) for transplantation. Dimethylsulphoxide (DMSO), the most commonly used cryoprotectant, is toxic to cells at higher concentrations (>10%); moreover, the freezing-thawing procedure itself is inevitably connected with the loss of HSPC. However, by chance we observed that short exposure of HSPC to DMSO enhances the responsiveness of these cells to an SDF-1 gradient and since SDF-1 is a major chemoattractant that navigates homing of HSPC to BM we became interested in elucidating this phenomenon. We found that short incubation (5–10 min) of human CB mononuclear cells (MNC) with DMSO at concentrations employed for cryopreservation (5–10%) significantly upregulates the expression of both CXCR4 (x 2–3) and CD34 (x 1.5) on CB MNC (as measured by FACS). Furthermore, DMSO significantly increased the chemotactic responsiveness (x 2–4) of CB MNC, BM MNC and selected CXCR4+ human hematopoietic cell lines (Jurkat, THP-1 cells) when the cells were exposed to 5–10% DMSO before chemotaxis assay. These responses to an SDF-1 gradient correlated with enhanced chemotaxis also of human CD34+, CD34+ CD38+, CD34+ CD38−, and CD34+ CXCR4+ clonogeneic progenitor cells, suggesting that DMSO directly enhances the responsiveness of human early progenitors (p<0.0001). At the molecular level, 5–10% DMSO strongly stimulated and prolonged SDF-1-dependent AKT phosphorylation. However, at the same time DMSO inhibited phosphorylation of MAPKp42/44. Similar observations were made for Sca-1+ BM-derived murine cells. In parallel experiments we found that murine Sca-1+ cells when preincubated with DMSO formed more 12 day-CFU-S colonies in spleens after transplantation into irradiated syngeneic recipients. Accordingly, x 2 more CFU-S were formed when Sca-1+ cells were exposed before transplantation to 5% DMSO and about x 4 more after exposure to 10% DMSO. Finally we employed a Ly5.1/Ly5.2 congeneic transplant model and showed that transplantation of Ly5.1 Sca-1+ cells exposed to 10% DMSO before transplantation resulted in higher chimerism in transplanted Ly5.2 mice as compared to untreated cells (control) (p<0.0001). In conclusion, we show for the first time an unexpected beneficial role of DMSO (5–10%) in regulation of homing of HSPC after transplantation and suggest that a short priming of HSPC with DMSO, even of non-cryopreserved cells, before transplantation may become a new strategy to enhance engraftment

Immortalization of Human Umbilical Cord Blood-Derived Hematopoietic Progenitor Cells.

Besides progressive telomere shortening, human cells undergo senescence in response to various types of stress. The stress-related signaling pathways triggered converge to varying extents on the p53 and retinoblastoma (Rb) tumor suppressors, and the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p16INK4a. We therefore attempted to extend the life-span of human CD34+ cord blood (CB) progenitors by ectopically expressing the human papillomavirus type 16 (HPV16) E6 and E7 genes, which accelerate the degradation of p53 and Rb, respectively, separately and in conjunction with the gene encoding the reverse transcriptase catalytic subunit of human telomerase (hTERT). CD34+ CB cells were transduced with VSV-G glycoprotein-pseudotyped self-inactivating lentiviral vectors that express HPV16 E6/E7 or hTERT linked to downstream GFP/YFP reporter genes on bicistronic transcripts. Sorted GFP+ and/or YFP+ CB cells were maintained under serum-free conditions in the presence of stem cell factor (100 ng/ml), Flt3 ligand (100 ng/ml), thrombopoietin (20 ng/ml) plus interleukin-3 (20 ng/ml). In all cases (n = 3), control CD34+ CB cells differentiated into macrophage-like cells and underwent senescence-associated proliferation arrest after ~4 months in culture. Constitutive expression of hTERT did not increase CB cell replicative capacity in repeated attempts (n = 3), and macrophage-like cells were also the predominant cell type that accumulated in these cultures. In contrast, CB cells constitutively expressing HPV16 E6/E7 alone (n = 2) or in concert with hTERT (n = 5) continued to proliferate beyond 4 months, eventually giving rise to permanent (> 2 years) cell lines with a CD45+CD34−CD44+CD235a+CD71+ CD203+CD33+CD13+ myeloerythroid/mast cell progenitor phenotype. Interestingly, two CB cell lines coexpressing E6/E7 plus hTERT were also positive for the CD133 hematopoietic stem/progenitor cell antigen. When the immortalized CB cells were cultured in the presence of erythropoietin, slight up-regulation of the CD36 erythroid marker was observed. Moreover, when selected CB cells were subjected to a myeloid differentiation regimen, up to 90% of the cells acquired the ability to adhere to fibronectin in the best example, of which ~30% were capable of superoxide-dependent nitroblue tetrazolium reduction reflective of terminal monocytic differentiation. Notably, CB cell cultures expressing only HPV16 E6/E7 went through a crisis period and, when analyzed by spectral karyotyping, G-banding and fluorescence in situ hybridization with centromere-specific probes, were found to be highly aneuploid. By comparison, the CB cell lines obtained by coexpression of HPV16 E6/E7 plus hTERT exhibited near diploid karyotypes with minimal chromosomal aberrations, concomitant with stabilization of telomere length. Upon injection of the two CD133+ E6/E7 plus hTERT-expressing CB cell lines subcutaneously (5 x 106 cells) or intravenously (1 x 107 cells) into sublethally-irradiated (250 cGy) NOD/SCID mice, no tumors developed after 7 months of observation (n = 11), with tumorigenic conversion requiring cooperative enforced expression of a v-H-ras or BCR-ABL oncogene (n = 12). These findings establish the feasibility of bypassing senescence in human hematopoietic progenitors through genetic engineering, providing proof of principle for gene-based or other approaches that might eventually allow establishment of permanent human hematopoietic stem cell lines.

Establishment and Characterization of a New Human Ph+ Erythroleukemic Cell Line, VES.

Continuous human malignant hematopoietic cell lines are invaluable tool for hematological research. Here we report on a new erythroleukemic cell line termed VES that was established from the bone marrow mononuclear cells (BMNC) of a 22-year-old woman with Ph+ CML during her second post-transplant period. Actually, after unsuccessful allo-transplantation, the patient received auto-transplant that also failed to engraft. At the time of initiation of BMNC into culture for the analysis of stromal cell capacity, her bone marrow was tested Ph-negative by both FISH and PCR. Instead of establishment of an adherent stromal layer in vitro, blast-like cells appeared in the supernatant on day 27. These blast cells expressed highly amplified bcr/abl locus as shown by FISH, whereas the patient's bone marrow was still Ph-negative at the time. Following a very short period, the cultured cells started to express stable features of erythroleukemia cell line.Optimal growth was obtained by suspending the cells at concentration of 0.3x106 cells/ml in IMDM containing 10% FBS, 1% penicillin-streptomycin and 1% L-glutamine solution. After 3 days of culture, cell concentration varied between 1.3 and 1.8x106 cells/ml. VES cells were tested negative for the presence of EBV virus. For analysis of clonogenicity, VES cells at concentration of 103/ml were grown in a serum-free methylcellulose medium without cytokines (H4236; StemCell Tech., Canada). Following 7-day culture, 103 VES cells produced 251 ±42 and after 14 days 431 ±30 GF-independent colonies. Furthermore, on day 14 of the cell culture it was possible to observe reddish color of the colonies indicating the presence of hemoglobin. Cytological examination of cytospin preparations indicated homogenous population of leukemia blasts (>80%) the majority of them (>90%) being glycophorinA positive and MPO negative. Imunophenotyping and multiparameter flow cytometry revealed proerythroblastic lineage-associated profile: GlyA/CD235a+, CD11b+, CD15+, CD29+, CD33+, and CD117+. Conventional cytogenetics revealed complex karyotype with multiple numerical/structural abnormalities (MAKA), while metaphase FISH revealed the following aberrations: t(9;22), 5q31, 7q31, +8, +6.Since FISH analysis detected highly amplified bcr/abl locus, we tested VES cells' sensitivity to imatinib (Gleevec). Treatment with imatinib for 3 days (MTS assay) inhibited proliferation of VES cells with IC50 value of 0.2mM. Following 14 days of culture in methylcellulose medium with addition of 0.2mM imatinib, the clonogenic potential of VES cells was reduced by 55% in relation to untreated control. We demonstrated that imatinib induced apoptosis in VES cells in time- and dose-dependant manner, assessed with AnnexinV and PI staining, while there were no significant changes observed in the cell cycle except for a mild increase in cells in G0/G1 phase. Although further analyses are required, we believe that VES cells represent a new Ph+ erythroleukemia cell line and a suitable model for studying Ph+ malignant hematopoiesis in vitro.

New Monoclonal Antibodies Reactive with Human Bone Marrow-Derived Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) could be well isolated and expanded from bone marrow and have been widely studied. In addition to hMSCs have ability to support hematopoiesis, they have generated a great deal of excitement as a potential source of cells for regenerative medicine and tissue engineering owing to their dramatic potential of proliferation and differentiation. However, the precise identity of hMSCs remains a challenge and the homogeneity of hMSCs is still controversial due in part to the lack of useful cell-specific markers. Thus, the objective of our study was to development monoclonal antibodies (MAbs) reactive with hMSCs surface antigens. hMSCs were isolated from bone marrow of volunteers on a density gradient solution by Ficoll-paque and cultured (LG-DMEM, 10%FBS) with their characteristic of adherence. hMSCs phenotype were analyzed by flow cytometry and demonstrated that they were uniformly positive for CD29, CD44, CD166 and CD105, negative for CD34, CD45 and HLA-DR. All hMSCs could be successfully induced to differentiate into osteogenic precursor cells, adipocytes and neuron cells when treated by correctly conditioned medium. There were detected to be no significant differences of phenotypes, growth characteristics and the potential of differentiation after either passage or programmed cryopreservation. Cultured and passaged hMSCs were used to immunized BALB/C mice at 1-week interval for 4 times, and their splenocytes subsequently harvested were fused with mouse SP 2/0 myeloma cells in the presence of PEG followed by culturing in the HAT selective medium. Indirect immunofluorescence assay (fluorescent antibody reacted with IgG and IgM isotypes) was utilized to preliminary screen the hybridoma colonies which synthesized and secreted antibodies having the specificity for cultured hMSCs. Out of 1248 wells initially plated, only 27 wells were tested positive for reactivity with living cultured hMSCs, and finally we got 5 hybridoma cell lines which could secreted MAbs steadily, named ZUB1, ZUB4, ZUC3, ZUE12, ZUF10. In order to identify hybridomas which secreted antibodies were only specific for hMSCs, peripheral blood cells, human malignant hematopoietic cell lines and rat marrow-derived mesenchymal stem cells were detected by indirect immunofluorescence analysis and subsequently a series of mesenchymal and non-mesenchymal derived tissues of human were performed by immunohistochemistry staining. The results demonstrated that all these 5 MAbs were specific for hMSCs except that ZUC3 had cross reaction with rat marrow-derived mesenchymal stem cells, and there were a few positive cells in the bone marrow. The subtype of monoclonal antibody ZUC3 was IgM, while other four MAbs were IgG1 and light chains of all MAbs were kappa. Using hybridoma cell lines, large amount of MAbs in ascites were generated then purified. MAbs ZUB1, ZUB4, ZUC3, ZUE12, ZUF10 were used to detected cultured hMSCs by flow cytometry, and percentage of positive cells are 87.39%, 88.07%, 88.12%, 69.89%, 83.67%, respectively. In conclusion, there 5 new MAbs that developed reactived with hMSCs surface antigens. Our study would further the understanding of hMSCs biological characterization and have potential application of new MAbs for identifying, sorting hMSCs derived from bone marrow. Further studies will focus on the characterization and epitopes of the MAbs to hMSCs.

Crystal Structure of the First KH Domain of Human Poly(C)-binding Protein-2 in Complex with a C-rich Strand of Human Telomeric DNA at 1.7 Å

Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs). To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-A resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.

Expression of Leptin Receptors in Mononuclear Cells from Myelodysplastic Syndromes and Acute Myeloid Leukemias

Leptin, the adipocyte hormone, and its receptor have been implicated in the differentiation/proliferation of hematopoietic cells. Given that the deregulated expression of a variety of growth factors and/or their receptors has been implicated in the pathogenesis of certain leukemias, we aimed to characterize the potential differences in the expression pattern of the two major leptin receptor transcript variants in peripheral blood mononuclear cells (PBMC) between different hematologic malignancies. Using RT-PCR and Southern blotting, we compared the expression levels of the two major leptin receptors, the longest (OB-R_L) and the shortest (OB-R_S) splice variants, in PBMC from patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), healthy individuals and two human hematopoietic cell lines (HL-60 and K562).Expression of the OB-R_S transcript clearly exceeded that of OB-R_L in all patients and controls and in the HL-60 cells, but this was reversed in the K562 cell line. However, the expression of the OB-R_L was significantly lower in MDS compared to controls and tended to be so in AML, while OB-R_S tended to be higher in MDS and AML patients compared to controls, but this difference was not significant.Serum leptin levels and circulating soluble leptin receptor levels were slightly but not significantlyhigher in AML and MDS. These alterations in the expression of the leptin receptor isoforms in MDS and AML patients could suggest a potential role of leptin and its signaling in hematopoietic malignancies, which requires further examination.

Mitochondrial Targeting of HumanO6-Methylguanine DNA Methyltransferase Protects against Cell Killing by Chemotherapeutic Alkylating Agents

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

Enhanced function with decreased internalization of carboxy-terminus truncated CXCR4 responsible for WHIM syndrome

WHIM (warts, hypogammaglobulinemia, recurrent bacterial infection, myelokathexis) syndrome is an autosomal dominant immune deficiency with severe chronic neutropenia and marrow neutrophil apoptosis. Carboxy-termini truncating mutations in the chemokine receptor CXCR4 have been identified in WHIM patients. We created a retrovirus encoding mutated CXCR4 (truncating point mutation 1000C-->T [R334X] inherited heterozygously in several WHIM patients) in order to transducer healthy human CD34 stem cells and K562 to overexpress mutated CXCR4 and determined its effect on receptor responses to stromal-derived factor-1 (SDF1). Retrovirus vector was engineered to coexpress WHIM-associated R334X mutated CXCR4 together with green fluorescent protein (GFP). Control vectors included similar constructs with wild-type CXCR4 (WT-CXCR4) or only GFP. CD34+ cells and K562 were transduced with these vectors. Populations of 100% transduced K562 were established by sorting GFP+ cells by flow cytometry. We performed migration and calcium flux assays of transduced CD34+ cells and transduced/sorted K562. We also examined receptor recycling in response to SDF1. Healthy human CD34+ cells and/or human erythroleukemia K562 cells transduced to express mutated CXCR4, WT-CXCR4, or GFP alone demonstrated that mutated CXCR4 was associated with enhanced calcium flux and enhanced migration. There was also decreased receptor internalization and enhanced recovery of surface mutated CXCR4 in response to SDF1 compared with WT-CXCR4. We propose that decreased internalization of WHIM-associated mutated CXCR4 leads to prolongation/enhancement of signaling in response to SDF1 and that this may provide the biochemical basis for the autosomal dominant abnormalities of cell trafficking and function associated with WHIM syndrome.

Optimization of equine infectious anemia derived vectors for hematopoietic cell lineage gene transfer

Gene transfer into hematopoietic cells may allow correction of a variety of hematopoietic and metabolic disorders. Optimized HIV-1 based lentiviral vectors have been developed for improved gene transfer and transgene expression into hematopoietic cells. However, the use of HIV-1 based vectors for human gene therapy may be limited due to ethical and biosafety issues. We report that vectors based on the non-primate equine infectious anemia virus (EIAV) transduce a variety of human hematopoietic cell lines and primary blood cells. To investigate optimization of gene expression in hematopoietic cells, we compared a variety of post-transcriptional elements and promoters in the context of EIAV vectors. We observed cell specific increase in the number of transgene expressing cells with the different post-transcriptional elements, whereas the use of elongation factor alpha 1 (EFalpha1) promoter resulted in significant increases in both the number of transgene expressing cells and the level of transgene protein in all cell types tested. We then demonstrate increased transduction of hematopoietic cells using a second-generation EIAV vector containing a self-inactivating EIAV LTR and the EIAV central polypurine tract (cppt). These data suggest that optimized EIAV vectors may be a suitable alternative to HIV-1 vectors for use in hematopoietic gene therapy.