DPP4 truncated GM-CSF and IL-3 manifest distinct receptor-binding and regulatory functions compared with their full-length forms.

Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony-stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated (T) factors (T-granulocyte-macrophage-CSF (T-GM-CSF)) and T-interleukin-3 (T-IL-3)) on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and -IL-3 had enhanced receptor binding, but decreased CSF activity, compared with their FL forms. Importantly, T-GM-CSF and -IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and -IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster-forming cells from patients with acute myeloid leukemia regardless of cytogenetic or molecular alterations and in vivo using animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared with their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.

Activation of OCT4 enhances ex vivo expansion of human cord blood hematopoietic stem and progenitor cells by regulating HOXB4 expression.

Although hematopoietic stem cells (HSC) are the best characterized and the most clinically used adult stem cells, efforts are still needed to understand how to best ex vivo expand these cells. Here we present our unexpected finding that OCT4 is involved in the enhancement of cytokine-induced expansion capabilities of human cord blood (CB) HSC. Activation of OCT4 by Oct4-activating compound 1 (OAC1) in CB CD34(+) cells enhanced ex vivo expansion of HSC, as determined by a rigorously defined set of markers for human HSC, and in vivo short-term and long-term repopulating ability in NSG mice. Limiting dilution analysis revealed that OAC1 treatment resulted in 3.5-fold increase in the number of SCID repopulating cells (SRCs) compared with that in day 0 uncultured CD34(+) cells and 6.3-fold increase compared with that in cells treated with control vehicle. Hematopoietic progenitor cells, as assessed by in vitro colony formation, were also enhanced. Furthermore, we showed that OAC1 treatment led to OCT4-mediated upregulation of HOXB4. Consistently, siRNA-mediated knockdown of HOXB4 expression suppressed effects of OAC1 on ex vivo expansion of HSC. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC.

Small-molecule inhibitors targeting the DNA-binding domain of STAT3 suppress tumor growth, metastasis and STAT3 target gene expression in vivo.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness. Thus targeting STAT3 promises to be an attractive strategy for treatment of advanced metastatic tumors. Although many STAT3 inhibitors targeting the SH2 domain have been reported, few have moved into clinical trials. Targeting the DNA-binding domain (DBD) of STAT3, however, has been avoided due to its 'undruggable' nature and potentially limited selectivity. In a previous study, we reported an improved in silico approach targeting the DBD of STAT3 that resulted in a small-molecule STAT3 inhibitor (inS3-54). Further studies, however, showed that inS3-54 has off-target effect although it is selective to STAT3 over STAT1. In this study, we describe an extensive structure and activity-guided hit optimization and mechanistic characterization effort, which led to identification of an improved lead compound (inS3-54A18) with increased specificity and pharmacological properties. InS3-54A18 not only binds directly to the DBD and inhibits the DNA-binding activity of STAT3 both in vitro and in situ but also effectively inhibits the constitutive and interleukin-6-stimulated expression of STAT3 downstream target genes. InS3-54A18 is completely soluble in an oral formulation and effectively inhibits lung xenograft tumor growth and metastasis with little adverse effect on animals. Thus inS3-54A18 may serve as a potential candidate for further development as anticancer therapeutics targeting the DBD of human STAT3 and DBD of transcription factors may not be 'undruggable' as previously thought.

Insights into the biology of cord blood stem/progenitor cells.

OBJECTIVES: To review information on cord blood banking and transplantation with respect to the author's studies, and in context of this field of investigation. RESULTS: Cord blood transplantation has been successfully used to treat a number of malignant and non-malignant disorders. However, this technique is still associated with limited numbers of cells for transplantation, and with delayed engraftment of neutrophils and platelets. The field of cord blood transplantation will benefit from enhanced and mechanistically based information on haematopoietic stem cell function and potential means to enhance its effectiveness are reviewed. This includes notions concerning possibility of retrieving more cells from the placenta and cord blood, to expand haematopoietic stem cells ex vivo and to increase efficiency of homing and engraftment of these cells. Also discussed are cryopreservation and long-term storage of cord blood haematopoietic and progenitor cells, and new laboratory findings and animal studies for non-haematopoietic uses of cord blood.

Experimental basis of cord blood transplantation.

Efforts are needed to enhance the efficacy of cord blood (CB) transplantation. Laboratory information set the stage for the first and subsequent CB transplants, and will be instrumental in continuing to advance the field. This paper offers a brief understanding of the current state of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) biology, a look back at laboratory studies leading to the first CB transplants, and a discussion of the possible means to enhance CB transplantation. Results show that physical recovery of greater numbers of HPCs is possible after CB is collected by perfusing the placenta, but how realistic this procedure is for collection of CB to be banked is open to question. We also show that the chemokine stromal cell-derived factor-1/CXCL12 can enhance the ex vivo expansion of CB HPCs beyond that of the combination of SCF, Flt3-ligand and TPO. Advances in cytokine and stromal cell biology, and in intracellular signals mediating the effects of cytokines/stromal cells should be considered in the context of future efforts to enhance functional activities of donor CB HSCs and HPCs and the microenvironmental niche of the recipient, which is required for acceptance and nurturing these HSCs/HPCs.

Negative regulation of chemokine receptor CXCR4 by tumor suppressor p53 in breast cancer cells: implications of p53 mutation or isoform expression on breast cancer cell invasion.

Chemokine receptor CXCR4 and its ligand CXCL12 are suggested to be involved in migration, invasion and metastasis of breast cancer cells. Mutation of the tumor suppressor gene p53 in breast cancer is associated with metastasis and aggressive clinical phenotype. In this report, we demonstrate that wild type but not the dominant-negative mutant (V143A) or cancer-specific mutants (R175H or R280K) of p53 repress CXCR4 expression. Recently described cancer-specific p53 isoform, Delta133p53, also failed to repress CXCR4 promoter activity. Short-interfering RNA-mediated depletion of p53 increased endogenous CXCR4 expression in MCF-7 breast cancer cells that contain wild-type p53. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 [HCT116(p53KO)] was 10-fold higher compared to that in parental HCT116 cells with functional wild-type p53. Deletion analysis of CXCR4 promoter identified a seven-base pair p53-repressor element homologous to cyclic AMP/AP-1 response (CRE/AP-1) element. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed binding of ATF-1 and cJun to the CRE/AP-1 element. The p53 rescue drug PRIMA-1 reduced CXCR4 mRNA and cell surface expression in MDA-MB-231 cells, which express R280K mutant p53. CP-31398, another p53 rescue drug, similarly reduced cell surface levels of CXCR4. PRIMA-1-mediated decrease in CXCR4 expression correlated with reduced invasion of MDA-MB-231 cells through matrigel. These results suggest a mechanism for elevated CXCR4 expression and metastasis of breast cancers with p53 mutations or isoform expression. We propose that p53 rescue drugs either alone or in combination with chemotherapeutic drugs may be effective in reducing CXCR4-mediated metastasis.

Biology of cord blood cells and future prospects for enhanced clinical benefit.

Cord blood (CB) has served as a clinically beneficial source of hematopoietic stem (HSC) and progenitor (HPC) cells for transplantation and correction of a large number of malignant and non-malignant disorders. The capacity of CB to perform these functions is intimately related to the quality and quantity of HSC and HPC present in CB. This review covers the biology of HSC and HPC, efforts to expand these cells ex vivo for enhanced clinical utility that has thus far not been very successful, and recent studies on attempts to enhance the homing and engrafting capability of HSC as an alternative means for more effective use of the limited numbers of CB cells collected. This review also highlights the presence in CB of mesenchymal stem cells, unrestricted somatic stem cells, endothelial progenitor cells and immune cells. The presence and biology of these non-HSC/HPC may open up future possibilities for additional clinical benefit of CB, a product considered mainly for discard before its clinical transplantation potential was realized in the late 1980s.

SDF-1alpha/CXCL12 enhances retroviral-mediated gene transfer into immature subsets of human and murine hematopoietic progenitor cells.

Genetic modification of hematopoietic stem and progenitor cells has the potential to treat diseases affecting blood cells. Oncoretroviral vectors have been used for gene therapy; however, clinical success has been limited in part by low gene transfer efficiencies. We found that the presence of stromal-derived factor 1 (SDF-1alpha)/CXCL12 during retroviral transduction significantly enhanced, in a dose-dependent fashion, gene transfer into immature subsets of high proliferative human and murine hematopoietic progenitor cells. Murine mononuclear bone marrow cells and purified c-Kit(+)Lin(-) bone marrow cells were prestimulated and transduced with the bicistronic retroviral vector MIEG3 on Retronectin-coated surfaces in the presence and absence of SDF-1. SDF-1 enhanced gene transduction of murine bone marrow and c-Kit(+)Lin(-) cells by 35 and 29%, respectively. Moreover, SDF-1 enhanced transduction of progenitors in these populations by 121 and 107%, respectively. SDF-1 also enhanced transduction of human immature subsets of high proliferative progenitors present in either nonadherent mononuclear or CD34(+) umbilical cord blood cells. Transduction of hematopoietic progenitors was further increased by preloading Retronectin-coated plates with retrovirus using low-speed centrifugation followed by increasing cell-virus interactions through brief centrifugation during the transduction procedure. These results may be of clinical relevance.

Role of the CC chemokine receptor 9/TECK interaction in apoptosis.

Chemokine receptors are members of the G protein coupled receptor (GPCR) supergene family whose expression is highly restricted to hematopoietic cells. Although the primary role of chemokine and chemokine receptor interaction is believed to be regulation of chemotaxis of leukocytes, subsequent information clearly suggests that multiple immune regulatory functions are attributed to chemokine receptor signaling. We recently showed that activation of the CC chemokine 9 receptor (CCR9), a thymus-specific chemokine receptor, led to potent cFLIP(L)-independent resistance to cycloheximide-induced apoptosis and modest resistance to Fas-mediated apoptosis possibly via activation of multiple signaling components involving Akt and glycogen synthase kinase 3beta. The fact that these two apoptotic signals involve activation of similar arrays of death execution machinery such as caspase-8, caspase-9, or caspase-3, suggests that chemokine receptor signaling may provide a wide range of antiapoptotic activities to hematopoietic cells under certain biological conditions. GPCR is a large family of cell surface receptors, many of which are critically involved in hormonal and behavioral control. Recent observations also suggest that GPCR signaling plays a pivotal role in immune cell activation. Heterotrimeric G protein is an integral part of GPCR signaling. Thus, dissection of signaling components involved in the CCR9-mediated antiapoptosis could be a framework for cell survival mechanisms and may provide options for therapeutic interventions for neurdegenerative diseases or T cell malfunctioning.

Steel factor regulates cell cycle asymmetry.

Asymmetric segregation of cell-fate determinants during mitosis (spatial asymmetry) is an essential mechanism by which stem cells are maintained while simultaneously giving rise to differentiated progenitors that ultimately produce all the specialized cells in the hematopoietic system. Temporal cell cycle asymmetry and heterogeneity are attributes of cell proliferation that are also essential for maintaining tissue organization. Hematopoietic stem cells (HSCs) are regulated by a complex network of cytokines, some of which have very specific effects, while others have very broad ranging effects on HSCs. Some cytokines, like steel factor (SLF), are known to synergize with other cytokines to produce rapid expansion of progenitor cells. Using the human growth factor-dependent MO7e cell line as a model for synergistic proliferation, we present evidence that links proliferation asymmetry to SLF synergy with GM-CSF, and suggests that temporal asymmetry and cell cycle heterogeneity can be regulated by SLF in vitro. We also show that CDK-inhibitor and cell cycle regulator, p27kip-1, may be involved in this temporal asymmetry regulation. We propose that SLF/GM-CSF synergy is, in part, due to a shift in proliferation pattern from a heterogeneous and asymmetric one to a more synchronous and symmetric pattern, thus contributing dramatically to the rapid expansion that accompanies SLF synergy observed in MO7e cells. This kinetic model of asymmetry is consistent with recent evidence showing that even though SLF synergy results in a strong proliferative signal, it does not increase primary HSC self-renewal, which is believed to be highly dependent on asymmetric divisions. The factor-dependent MO7e/SCF- synergy/asymmetry model described here may therefore be useful for studies of the effects of various cytokines on cell cycle asymmetry.

Cutting edge: IL-17F, a novel cytokine selectively expressed in activated T cells and monocytes, regulates angiogenesis and endothelial cell cytokine production.

A novel secreted cytokine, termed IL-17F, was cloned using nested RACE PCR. This cytokine bears homology to IL-17. IL-17F was expressed only in activated CD4(+) T cells and activated monocytes. Recombinant human IL-17F did not stimulate the proliferation of hematopoietic progenitors or the migration of mature leukocytes. However, it markedly inhibited the angiogenesis of human endothelial cells and induced endothelial cells to produce IL-2, TGF-beta, and monocyte chemoattractant protein-1.

Regulation of hematopoiesis by chemokine family members.

Chemokines, originally designated as chemoattractant cytokines, comprise a large family of molecules that have been implicated in a number of different functions mediated through chemokine receptors. Among these functions are regulatory roles in hematopoiesis that encompass effects on the proliferation, survival, and homing/migration of myeloid progenitor cells. This article reviews the field of chemokine regulation of hematopoiesis at the level of myeloid progenitor cells.

Blocking of c-FLIP(L)--independent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction.

Chemokines play a pivotal role in regulating leukocyte migration as well as other biological functions. CC chemokine receptor 9 (CCR9) is a specific receptor for thymus-expressed CC chemokine (TECK). It is shown here that engagement of CCR9 with TECK leads to phosphorylation of Akt (protein kinase B), mitogen-activated protein kinases (MAPKs), glycogen synthase kinase--3 beta (GSK-3 beta), and a forkhead transcription factor, FKHR, in a human T-cell line, MOLT4, that naturally expresses CCR9. By means of chemical inhibitors, it is shown that phosphoinositide-3 kinase (PI-3 kinase), but not MAPK, is required for CCR9-mediated chemotaxis. Akt, GSK-3 beta, FKHR, and MAPK have been previously implicated in cell survival signals in response to an array of death stimuli. When MOLT4 cells, which expressed Fas as well as CXCR4, were stimulated with cycloheximide (CHX), an agonistic anti-Fas antibody, or a combination of these, the cells rapidly underwent apoptosis. However, costimulation of MOLT4 cells with TECK or stromal derived factor--1 significantly blocked CHX-mediated apoptosis, whereas stimulation only with TECK partially blocked Fas-mediated apoptosis. Concomitant with this blocking, cleavage of poly (adenosine 5'-diphosphate--ribose) polymerase and activation of caspase 3 were significantly attenuated, but the expression level of FLICE inhibitory protein c-FLIP(L), which had been shown to be regulated by CHX, was unchanged. This demonstrates that activation of CCR9 leads to phosphorylation of GSK-3 beta and FKHR and provides a cell survival signal to the receptor expressing cells against CHX. It also suggests the existence of a novel pathway leading to CHX-induced apoptosis independently of c-FLIP(L). (Blood. 2001;98:925-933)

Therapeutic potential of 4-1BB (CD137) as a regulator for effector CD8(+) T cells.

A fundamental problem of antitumor immunity is tumor-induced immunosuppression. Tumor cells often down-regulate expression of co-stimulatory molecules, tumor antigens, and major histocompatibility complex (MHC) molecules on tumor cells, secrete immunosuppressive substance such as transforming growth factor-beta (TGF-beta) or interleukin-4 (IL-4), and induce apoptosis of effector T cells to escape surveillance. A major goal of antitumor or antivirus immunotherapy is to generate long-lived protective T cells that enable killing of target cells. In this review, we discuss the importance of 4-1BB for development or survival of functionally active effector CD8(+) T cells against tumors, virus infection, and allogeneic immune responses and for potential therapeutic application.

Mzf1 controls cell proliferation and tumorigenesis.

MZF1 is a transcription factor belonging to the Krüppel family of zinc finger proteins, expressed in totipotent hemopoietic cells as well as in myeloid progenitors. Here we have inactivated Mzfi1 by gene targeting. Mzf1(-/-) mice develop lethal neoplasias characterized by the infiltration and complete disruption of the liver architecture by a monomorphic population of cells of myeloid origin reminiscent of human chloromas. Mzf1 inactivation results in a striking increase of the autonomous cell proliferation and of the ability of Mzf1(-/-) hemopoietic progenitors to sustain long-term hemopoiesis. These findings demonstrate that Mzf1 can act as a tumor/growth suppressor in the hemopoietic compartment.

Chemokine regulation of hematopoiesis and the involvement of pertussis toxin-sensitive G alpha i proteins.

Chemokines have been implicated in regulation of various aspects of hematopoiesis, including negative regulation of the proliferation of immature subsets of myeloid progenitor cells (MPCs), chemotaxis of MPCs, and survival enhancement of MPCs after delayed growth factor addition. Since chemokine receptors are seven-transmembrane-spanning G-protein-linked receptors and the chemotactic effect in vitro of the CXC chemokine SDF-1 is pertussis toxin (PT)-sensitive, implying the involvement of G alpha i proteins as mediators of SDF-1-induced chemotaxis, we evaluated the effects of PT on other chemokine actions influencing MPCs. While the in vitro survival-enhancing effects of SDF-1 on GM-CSF and steel factor-dependent mouse bone marrow granulocyte macrophage progenitors (CFU-GM) were pertussis toxin-sensitive, the suppressive effects of the CC chemokine MIP-1 alpha and the CXC chemokine IL-8 on colony formation by GM-CSF and steel factor-sensitive CFU-GM were insensitive to pertussis toxin. These results suggest that not all chemokine-mediated effects on MPCs are necessarily mediated through pertussis toxin-sensitive G alpha i proteins.

Synergistic activation of RSK correlates with c-fos induction in MO7e cells stimulated with GM-CSF plus Steel factor.

Steel factor (SLF) plus GM-CSF induces proliferative synergy in factor-dependent cell line MO7e and hematopoietic progenitor cells. We previously reported ERK1/2 involvement in this synergy, but its downstream signaling molecules are not defined. Here, we investigated activation of the 90-kDa ribosomal S6 kinase (RSK) proteins by measuring the phosphorylation status and in vitro kinase activity in MO7e cells. Both GM-CSF and SLF induced activation of RSK, and the combined stimulation with these two cytokines induced synergistic and persistent activation of RSK. RSK activity was reduced by PI3 kinase inhibitor LY294002 or MEK1 inhibitor PD98059, suggesting that the ERK as well as the PI3 kinase pathways are involved in regulation of RSK activity. Sensitivities of RSK activity to inhibitory drugs correlated well with those of c-fos gene induction. Taken together, synergistic activation of RSK may contribute, at least in part, to the synergistic induction of c-fos after combined stimulation with GM-CSF plus SLF.

Human recombinant interferon-inducible protein-10: intact disulfide bridges are not required for inhibition of hematopoietic progenitors and chemotaxis of T lymphocytes and monocytes.

Human recombinant interferon-inducible protein-10 (rIP-10), a C-X-C chemokine, inhibits proliferation of human hematopoietic progenitors responsive to co-stimulation by recombinant steel factor (rSLF), is chemotactic for human monocytes and T-lymphocytes, and promotes T-lymphocyte adhesion to endothelial cells. Because chemokines have four conserved cysteines forming two intramolecular disulfide bridges, we decided to investigate their contribution in the biological activity of rIP-10. Since amino acid residues 22-98 of the sequence predicted by the cDNA constitute the naturally occurring IP-10, they were cloned after an initiating methionine into expression vector pET-3d. Subsequently rIP-10 was purified by enzymatic cell lysis, solubilization of refractile bodies with guanidine hydrochloride, renaturation by dialysis against dilute acetic acid, and sequential ion-exchange and reverse-phase high-performance liquid chromatography. Purified rIP-10 was reduced with 20 mM dithiothreitol, and chemically modified with 100 mM iodoacetamide (IAA), or S-methyl-methanethiosulfonate (MMTS), or N-methylmaleimide (NMM). Radiolabeling experiments demonstrated that 95% of the rIP-10 thiols were modified, and this was confirmed with SDS-PAGE. The biological activity of modified rIP-10 was determined in vitro by inhibition of rSLF-responsive human bone marrow hematopoietic progenitor proliferation and by chemotaxis assays using human T-lymphocytes and monocytes. In both assay systems, the biological activity was evident at rIP-10 concentrations of 20-100 ng/ml. The activity was preserved after modification of rIP-10 by IAA or MMTS, but was abolished after modification by NMM. We conclude that disulfide bridges are not essential for the biological activity of rIP-10.