Design, synthesis, and biological characterization of bivalent 1-methyl-1,2,5,6-tetrahydropyridyl-1,2,5-thiadiazole derivatives as selective muscarinic agonists.

Selective muscarinic agonists could be useful in the treatment of neurological disorders such as Alzheimer's disease, schizophrenia, and chronic pain. Many muscarinic agonists have been developed, yet most exhibit at best limited functional selectivity for a given receptor subtype perhaps because of the high degree of sequence homology within the putative binding site, which appears to be buried within the transmembrane domains. Bivalent compounds containing essentially two agonist pharmacophores within the same molecule were synthesized and tested for receptor binding affinity and muscarinic agonist activity. A series of bis-1,2,5-thiadiazole derivatives of 1,2,5,6-tetrahydropyridine linked by an alkyloxy moiety exhibited very high affinity (K(i) < 1 nM) and strong agonist activity. The degree of activity depended on the length of the linking alkyl group, which could be replaced by a poly(ethylene glycol) moiety, resulting in improved water solubility, binding affinity, and agonist potency.

Increased growth promoting but not mast cell degranulation potential of a covalent dimer of c-Kit ligand.

The native form of soluble c-kit ligand (KL) is a noncovalent dimer. We have isolated a soluble, disulfide-linked dimer of murine KL (KL-CD) by expressing KL in Escherichia coli and refolding the denatured protein under conditions that promote the formation of both noncovalent dimers (KL-NC) and KL-CD. KL-CD exhibits a 10- to 15-fold increase in the ability to stimulate the growth of both the human megakaryocytic cell line MO7e and murine bone marrow-derived mast cells relative to KL-NC. Colony-forming assays of murine bone marrow progenitor cells also reflected this increased potency. However, KL-CD and KL-NC are equally able to prime mast cells for enhanced IgE-dependent degranulation in vitro and activate mast cells in vivo. Improving the growth-promoting activity of KL without changing its mast cell activation potential suggests that KL-CD or a related molecule could be administered in the clinic at doses that stimulate hematopoietic recovery while avoiding significant mast cell activation.

Differential expression and processing of two cell associated forms of the kit-ligand: KL-1 and KL-2.

The c-kit ligand, KL, and its receptor, the proto-oncogene c-kit are encoded, respectively, at the steel (Sl) and white spotting (W) loci of the mouse. Both Sl and W mutations affect cellular targets in melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. Although identified as a soluble protein, the predicted amino acid sequence of KL indicates that it is an integral transmembrane protein. We have investigated the relationship between the soluble and the cell associated forms of KL and the regulation of their expression. We show that the soluble form of KL is generated by efficient proteolytic cleavage from a transmembrane precursor, KL-1. An alternatively spliced version of KL-1, KL-2, in which the major proteolytic cleavage site is removed by splicing, is shown to produce a soluble biologically active form of KL as well, although with somewhat diminished efficiency. The protein kinase C inducer phorbol 12-myristate 13-acetate and the calcium ionophore A23187 were shown to induce the cleavage of both KL-1 and KL-2 at similar rates, suggesting that this process can be regulated differentially. Furthermore, proteolytic processing of both the KL-1 and KL-2 transmembrane protein products was shown to occur on the cell surface. The relative abundance of KL-1 and KL-2 is controlled in a tissue-specific manner. Sld, a viable steel allele, is shown to encode a biologically active secreted mutant KL protein. These results indicate an important function for both the soluble and the cell associate form of KL. The respective roles of the soluble and cell associated forms of KL in the proliferative and migratory functions of c-kit are discussed.

Monoclonal antibody YB5.B8 identifies the human c-kit protein product.

The c-kit proto-oncogene encodes a 145- to 160-Kd transmembrane tyrosine kinase, which is a member of the platelet-derived growth factor receptor family and is allelic with the murine white spotting locus (W). W mutations affect several aspects of hematopoiesis, most notably erythroid progenitors and mast cells. A monoclonal antibody, YB5.B8, had been raised against the leukemic blasts of a patient with M1-type acute myelocytic leukemia (AML) and it precipitates a 150-Kd cell surface glycoprotein from leukemic cells. The YB5.B8 epitope is expressed on mast cells, on up to 3% of normal mononuclear bone marrow cells, and it identifies a sub-group of AML patients with a poor prognosis. In view of similarities noted between the cell surface antigen identified by YB5.B8 and the c-kit protein product, we performed experiments to determine whether they are identical. c-kit RNA expression in the cell lines HEL (human erythroleukemia) and A172 (glioblastoma) was shown to parallel the expression of the YB5.B8 epitope in these lines as measured by flow cytometry. Immunoprecipitation analysis with anti-kit serum and YB5.B8 antibody indicated that the two antibodies identified proteins of identical size in HEL (155 Kd) and A172 (145 Kd) cells, and sequential immunoprecipitations with the kit and the YB5.B8 antibodies demonstrated that the two antibodies recognize the same molecule. The proteins identified by both the anti-kit and YB5.B8 antibodies displayed in vitro autophosphorylation activity in immune complex kinase assays. In addition, YB5.B8 was able to inhibit the binding of the kit ligand to HEL cells. These studies provide evidence that the YB5.B8 antigen and the c-kit protein product are identical and raise certain hypotheses regarding the role of c-kit in AML.

The role of marrow accessory cell populations in the augmentation of human erythroid progenitor cell (BFU-E) proliferation by prostaglandin E.

The requirement for CD8+ T lymphocytes in the stimulation of erythroid progenitor cells by prostaglandin E (PGE) was examined. When low density bone marrow (LD-BM) or non-adherent bone marrow (NA-BM) cells were depleted of CD8+ cells the enhancing effect of PGE on BFU-E proliferation was abrogated. However, further enrichment of marrow progenitor cells by depletion of accessory cells using a cocktail of specific monoclonal antibodies, immunoadherence and fluorescence activated cell sorting with the MY10 monoclonal antibody resulted in a population of erythroid progenitor cells which were responsive to the enhancing effect of PGE despite the absence of CD8+ cells. Stepwise individual cell lineage depletion of marrow cell populations indicated that prostaglandin E enhanced erythroid burst formation in the absence of CD8+ cells provided that glycophorin-A+ cells were removed from LD-BM or NA-BM cells. These results suggest that nucleated erythroid cell populations may modulate the enhancement of BFU-E by PGE. The ability of GP-A+ cells to block the enhancement of erythroid burst formation by PGE following removal of CD8+ T cells was confirmed by readdition of conditioned medium prepared from positively selected GP-A+ marrow cells. These results expand the role of CD8+ T cells in the PGE enhancement of BFU-E proliferation and suggest another mechanism by which accessory cells regulate the proliferation of BFU-E in bone marrow.

Differential expression of class II MHC antigens in subpopulations of human hematopoietic progenitor cells.

Expression of major histocompatibility complex class II Ags HLA-DR, HLA-DP, and HLA-DQ on human BM granulocyte-erythroid-macrophage-megakaryocyte CFU (CFU-GEMM), BFU-E, and CFU-GM was examined by indirect immunofluorescence, cell sorting, and complement-mediated cytotoxicity. BM, highly enriched for progenitor cells by depletion of mature hematopoietic elements, was further separated by sterile sorting into HLA-DR (-), low, intermediate, and high intensity HLA-DR (+), as well as HLA-DP (+) and HLA-DP (-) cell fractions and assayed for progenitor cell content. In addition, in the case of HLA-DR, CFU-GM response to inhibition by prostaglandin E was determined. Cell sorting and cytotoxicity data confirm that approximately 95% of assayable erythroid, myeloid, and multipotential progenitor cells expressed HLA-DR, whereas HLA-DQ Ags were undetectable. HLA-DR and HLA-DP Ags were co-expressed on 61% of these progenitor cells, predominantly those expressing HLA-DR at high intensity. Day 7 and 14 CFU-GM showed a trend toward segregation to the high HLA-DR (+) cell fractions, especially when recombinant human G-CSF was used to stimulate clone formation. Both day 7 and day 14 CFU-GMs were found predominantly in the HLA-DP (+) cell fraction. In contrast, BFU-E and CFU-GEMM were found in the low intensity HLA-DR cell fraction and predominantly in the HLA-DP (-) fraction. Both eosinophil CFU and cells giving rise to basophil/mast cells in suspension culture were found in the low and intermediate intensity HLA-DR fractions, but could be segregated into HLA-DP (+) and HLA-DP (-) cell fractions, respectively. Functional analysis of day 7 CFU-GM segregated, based upon HLA-DR intensity, indicated a positive correlation between increasing HLA-DR intensity and responsiveness to inhibition by prostaglandin E. Furthermore, only those CFU-GM expressing HLA-DR at high intensity could be removed by cytolytic treatment using a mAb anti-HLA-DR previously shown to be selective for CFU-GM responsive to PGE and in S phase of the cell cycle.

Cell cycle specific effects of deferoxamine on human and murine hematopoietic progenitor cells.

The effect of the iron chelator deferoxamine (DSF) on the proliferation of normal erythroid and granulocyte-macrophage progenitor cells from human and murine bone marrow was examined. The addition of DSF at a concentration equivalent to the concentration of iron present in the culture system resulted in dose dependent inhibition of colony formation by human and murine granulocyte-macrophage progenitor cells and human normal erythroid progenitor cells. The addition of FeCl3 at culture initiation completely reversed the effects of DSF. Furthermore, significant numbers of progenitor cells could be rescued from the effects of DSF by iron added back as late as 24-48 h after exposure to DSF. The cell cycle specificity of DSF was also examined using bone marrow cells treated with high specific activity tritiated thymidine. Kinetic experiments demonstrated that in the presence of DSF the number of erythroid or granulocyte-macrophage colonies that could be rescued was dependent on the length of exposure to DSF. Comparisons between control and tritiated thymidine treated cells indicated that the proliferation of progenitor cells in S phase of the cell cycle was inhibited if iron was withheld until 6 and 24 h after exposure to DSF for murine and human cells, respectively, with little to no effect observed on progenitor cells not in S phase during this time period. These results confirm the importance of iron for hematopoietic progenitor cell proliferation and represent a new method by which the proliferation of cycling cells may be investigated in situ in semisolid culture systems.

Constitutive c-myc expression enhances the response of murine mast cells to IL-3, but does not eliminate their requirement for growth factors.

An interleukin-3 (IL-3) dependent mast cell line (MC) was infected with a recombinant retrovirus expressing the proto-oncogene c-myc and the drug selectable marker neo. Cells containing the transcriptionally activated c-myc gene displayed an increased growth rate in liquid culture and a higher cloning efficiency in soft agar when compared to control virus infected cells. All infected cells remained absolutely dependent on IL-3 for growth and were not tumorigenic in nude mice. Similar results were obtained with two additional IL-3 dependent cell lines, the mast cell 32D and the pre-B-cell Ea3. Thus, while constitutive expression of c-myc potentiates the response of mast cells to IL-3, it is not sufficient to eliminate their requirement for growth factors.

Synergistic inhibition of human marrow granulocyte-macrophage progenitor cells by prostaglandin E and recombinant interferon-alpha, -beta, and -gamma and an effect mediated by tumor necrosis factor.

The effects of prostaglandin E (PGE) and recombinant human interferon-alpha, -beta, and -gamma alone and in combination were tested for their effects on the proliferation of human bone marrow granulocyte-macrophage colony-forming units (GM-CFU). When tested alone, both classes of cytokines inhibited GM-CFU proliferation. In combination, PGE and all three types of recombinant interferons synergized in their ability to inhibit GM-CFU proliferation. Progressive enrichment for marrow GM-CFU indicated that the synergistic effects of PGE and interferon were dependent upon the presence of marrow-adherent cells. Studies using conditioned media from marrow-adherent cells prepared in the presence of interferon-alpha, -beta, and -gamma indicated that adherent cells produced a soluble factor in the presence of interferons that subsequently synergized with PGE in inhibiting GM-CFU proliferation. Neutralization of this conditioned media with a monoclonal antibody to tumor necrosis factor abrogated the synergistic inhibition of GM-CFU observed in the presence of PGE. The addition of recombinant tumor necrosis factor and PGE to accessory cell-depleted bone marrow resulted in synergystic inhibition of GM-CFU proliferation.