Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain

Tyrosine-specific phosphorylation of proteins is a key to the control of diverse pathways leading to cell growth and differentiation. The protein-tyrosine kinases described to date are either transmembrane proteins having an extracellular ligand binding domain or cytoplasmic proteins related to the v-src oncogene. Most of these proteins are expressed in a wide variety of cells and tissues; few are tissue-specific. Previous studies have suggested that lymphokines could mediate haematopoietic cell survival through their action on glucose transport, regulated in some cells through the protein-tyrosine kinase activity of the insulin receptor. We have investigated the possibility that insulin receptor-like genes are expressed specifically in haematopoietic cells. Using the insulin receptor-related avian sarcoma oncogene v-ros as a probe, we have isolated and characterized the complementary DNA of a novel gene, ltk (leukocyte tyrosine kinase). The ltk gene is expressed mainly in leukocytes, is related to several tyrosine kinase receptor genes of the insulin receptor family and has unique structural properties: it apparently encodes a transmembrane protein devoid of an extracellular domain. Two candidate ltk proteins have been identified with antibodies in the mouse thymus, and have properties indicating that they are integral membrane proteins. These features suggest that ltk could be a signal transduction subunit for one or several of the haematopoietic receptors.     

The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene

Mice homozygous for the recessive mutation osteopetrosis (op) on chromosome 3 have a restricted capacity for bone remodelling, and are severely deficient in mature macrophages and osteoclasts. Both cell populations originate from a common haemopoietic progenitor. As op/op mice are not cured by transplants of normal bone marrow cells, the defects in op/op mice may be associated with an abnormal haematopoietic microenvironment rather than with an intrinsic defect in haematopoietic progenitors. To investigate the molecular and biochemical basis of the defects caused by the op mutation, we established primary fibroblast cell lines from op/op mice and tested the ability of these cell lines to support the proliferation of macrophage progenitors. We show that op/op fibroblasts are defective in production of functional macrophage colony-stimulating factor (M-CSF), although its messenger RNA (Csfm mRNA) is present at normal levels. This defect in M-CSF production and the recent mapping of the Csfm structural gene near op on chromosome 3 suggest that op is a mutation within the Csfm gene itself. We have sequenced Csfm complementary DNA prepared from op/op fibroblasts and found a single base pair insertion in the coding region of the Csfm gene that generates a stop codon 21 base pairs downstream. Thus, the op mutation is within the Csfm coding region and we conclude that the pathological changes in this mutant result from the absence of M-CSF.     

The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus

Mice carrying mutations at the W locus located on chromosome 5 are characterized by severe macrocytic anaemia, lack of hair pigmentation and sterility. Mutations at this locus appear to affect the proliferation and/or migration of cells during early embryogenesis and result in an intrinsic defect in the haematopoietic stem cell hierarchy. An understanding of the molecular basis of the complex and pleiotropic phenotype in W mutant mice would thus provide insights into the important developmental processes of gametogenesis, melanogenesis and haematopoiesis. Here we show that the mouse mutant W has a deletion of the c-kit proto-oncogene. Interspecific backcross analysis demonstrates that the W locus is very tightly linked to c-kit and that the two loci cannot be segregated at this level of analysis. c-kit is the cellular homologue of the oncogene v-kit of the HZ4 feline sarcoma virus and encodes a transmembrane protein tyrosine kinase receptor that is structurally similar to the receptors for colony-stimulating factor-1 (CSF-1) and platelet derived growth factor. The co-localization of c-kit with W provides a molecular entry into this important region of the mouse genome. In addition, these observations provide the first example of a germ-line mutation in a mammalian proto-oncogene and implicate the c-kit gene as a candidate for the W locus.     

Clonal origin of haematopoietic colonies in the postnatal mouse liver

The liver of the neonatal mouse continues to show haematopoietic activity for up to 2 weeks after birth and morphological analysis has shown that this activity becomes focused in discrete haematopoietic colonies by the end of the first week postnatal. Furthermore, each colony contains cells of one haematopoietic lineage only, that is, erythroid, myeloid or pre-B-lymphoid cells. This pattern of differentiation suggests that each colony is derived from a single committed precursor cell, which, if true, would represent the first demonstration of non-mixed haematopoietic colonies in normal development and would provide a useful system for studying the factors affecting the clonal diversity of haematopoietic stem cells and their lineage-committed progeny. Here we have analysed the haematopoietic foci in the liver of neonatal mouse chimaeras, using a newly developed ubiquitous in situ cell marker system which clearly demonstrates the clonal origin of these colonies.     

Activating mutations in ALK provide a therapeutic target in neuroblastoma

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.     

Dynamics of chronic myeloid leukaemia

The clinical success of the ABL tyrosine kinase inhibitor imatinib in chronic myeloid leukaemia (CML) serves as a model for molecularly targeted therapy of cancer, but at least two critical questions remain. Can imatinib eradicate leukaemic stem cells? What are the dynamics of relapse due to imatinib resistance, which is caused by mutations in the ABL kinase domain? The precise understanding of how imatinib exerts its therapeutic effect in CML and the ability to measure disease burden by quantitative polymerase chain reaction provide an opportunity to develop a mathematical approach. We find that a four-compartment model, based on the known biology of haematopoietic differentiation, can explain the kinetics of the molecular response to imatinib in a 169-patient data set. Successful therapy leads to a biphasic exponential decline of leukaemic cells. The first slope of 0.05 per day represents the turnover rate of differentiated leukaemic cells, while the second slope of 0.008 per day represents the turnover rate of leukaemic progenitors. The model suggests that imatinib is a potent inhibitor of the production of differentiated leukaemic cells, but does not deplete leukaemic stem cells. We calculate the probability of developing imatinib resistance mutations and estimate the time until detection of resistance. Our model provides the first quantitative insights into the in vivo kinetics of a human cancer.     

The earliest thymic progenitors for T cells possess myeloid lineage potential

There exists controversy over the nature of haematopoietic progenitors of T cells. Most T cells develop in the thymus, but the lineage potential of thymus-colonizing progenitors is unknown. One approach to resolving this question is to determine the lineage potentials of the earliest thymic progenitors (ETPs). Previous work has shown that ETPs possess T and natural killer lymphoid potentials, and rare subsets of ETPs also possess B lymphoid potential, suggesting an origin from lymphoid-restricted progenitor cells. However, whether ETPs also possess myeloid potential is unknown. Here we show that nearly all ETPs in adult mice possess both T and myeloid potential in clonal assays. The existence of progenitors possessing T and myeloid potential within the thymus is incompatible with the current dominant model of haematopoiesis, in which T cells are proposed to arise from lymphoid-. Our results indicate that alternative models for lineage commitment during haematopoiesis must be considered.     

Epidermal growth factor-dependent phosphorylation of lipocortin

Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein.     

Stimulation of multipotential, erythroid and other murine haematopoietic progenitor cells by adherent cell lines in the absence of detectable multi-CSF (IL-3)

It is well established that murine multipotential and committed erythroid progenitor cells require the presence of a glycoprotein, termed multi-CSF (multi-colony-stimulating factor, IL-3) for clonal proliferation and differentiation in vitro. The initial proliferation of these cells can also be stimulated by two other glycoproteins, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), although continued proliferation and differentiation requires the subsequent presence of multi-CSF. Here we report the stimulation of multipotential, erythroid and other haematopoietic progenitor cells by a number of adherent cell lines including a cloned bone marrow cell line (B.Ad). The positive cell lines, as feeder layers, exhibit colony-stimulating, erythropoietin-like and burst-promoting (BPA) activities. Optimal erythropoietic stimulation by the B.Ad line requires close cell-cell contact. The cell lines also support the in vitro clonal growth of multipotential colony-forming cells and progenitors of six other haematopoietic lineages. The biological activities observed seem not to be mediated by known multipotential or erythroid colony-stimulating factors (multi-CSF, IL-3, MCGF, HCGF, PSF, BPA).     

VEGFR-2酪氨酸激酶抑制剂的三维定量构效关系研究

 运用比较分子力场分析对28个氨基吡唑并吡啶二芳基脲类VEGFR-2酪氨酸激酶抑制剂进行了三维定量构效关系研究。所得CoMFA模型交叉验证系数q2=0.681,回归系数r2=0.958,统计方差比F=64.964,影响药效的立体场和静电场的贡献分别为65.7％和34.3％。CoMFA模型的三维等值图为化合物的结构改造提供了参考。     

Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture.

Despite the importance of germ cells to the survival of species, surprisingly little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. Mutations in the murine Dominant White Spotting (W) and Steel genes, which respectively encode the c-kit tyrosine kinase receptor and the c-kit ligand (or Steel factor), impair the development of primordial germ cells (PGCs) in vivo, as well as haematopoietic stem cells and neural crest-derived melanoblasts. Here we use a monoclonal antibody against c-kit tyrosine kinase receptor and recombinant Steel factor to study the c-kit receptor-ligand system in cultured PGCs. In addition, we show that leukaemia inhibitory factor (also known as differentiation inhibitory activity), a factor secreted by STO fibroblasts, can stimulate proliferation of primordial germ cells in vitro.     

Adult T-cell progenitors retain myeloid potential

During haematopoiesis, pluripotent haematopoietic stem cells are sequentially restricted to give rise to a variety of lineage-committed progenitors. The classical model of haematopoiesis postulates that, in the first step of differentiation, the stem cell generates common myelo-erythroid progenitors and common lymphoid progenitors (CLPs). However, our previous studies in fetal mice showed that myeloid potential persists even as the lineage branches segregate towards T and B cells. We therefore proposed the 'myeloid-based' model of haematopoiesis, in which the stem cell initially generates common myelo-erythroid progenitors and common myelo-lymphoid progenitors. T-cell and B-cell progenitors subsequently arise from common myelo-lymphoid progenitors through myeloid-T and myeloid-B stages, respectively. However, it has been unclear whether this myeloid-based model is also valid for adult haematopoiesis. Here we provide clonal evidence that the early cell populations in the adult thymus contain progenitors that have lost the potential to generate B cells but retain substantial macrophage potential as well as T-cell, natural killer (NK)-cell and dendritic-cell potential. We also show that such T-cell progenitors can give rise to macrophages in the thymic environment in vivo. Our findings argue against the classical dichotomy model in which T cells are derived from CLPs; instead, they support the validity of the myeloid-based model for both adult and fetal haematopoiesis.     

Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation.

Increasing evidence indicates that the integrin family of cell adhesion receptors can transduce biochemical signals from the extracellular matrix to the cell interior to modulate cell growth and differentiation. We have shown that integrin/ligand interactions can trigger tyrosine phosphorylation of a protein of M(r) 120,000 (pp120), so it is possible that signal transduction by integrins might involve activation of intracellular protein tyrosine kinases as an early event in cell binding to the extracellular matrix. Here we report that pp120 is identical to the focal adhesion-associated protein tyrosine kinase pp125FAK (refs 3, 4). We show that tyrosine phosphorylation of this protein is modulated both by cell adhesion and transformation by pp60v-src, and that these changes in phosphorylation are correlated with increased pp125FAK tyrosine kinase activity. A model is proposed to relate these findings to the molecular basis of anchorage-independent growth of transformed cells.     

Haemangioblast commitment is initiated in the primitive streak of the mouse embryo

Haematopoietic and vascular cells are thought to arise from a common progenitor called the haemangioblast. Support for this concept has been provided by embryonic stem (ES) cell differentiation studies that identified the blast colony-forming cell (BL-CFC), a progenitor with both haematopoietic and vascular potential. Using conditions that support the growth of BL-CFCs, we identify comparable progenitors that can form blast cell colonies (displaying haematopoietic and vascular potential) in gastrulating mouse embryos. Cell mixing and limiting dilution analyses provide evidence that these colonies are clonal, indicating that they develop from a progenitor with haemangioblast potential. Embryo-derived haemangioblasts are first detected at the mid-streak stage of gastrulation and peak in number during the neural plate stage. Analysis of embryos carrying complementary DNA of the green fluorescent protein targeted to the brachyury locus demonstrates that the haemangioblast is a subpopulation of mesoderm that co-expresses brachyury (also known as T) and Flk-1 (also known as Kdr). Detailed mapping studies reveal that haemangioblasts are found at highest frequency in the posterior region of the primitive streak, indicating that initial stages of haematopoietic and vascular commitment occur before blood island development in the yolk sac.     

IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.     

Molecular characterization and expression of the gene encoding human erythroid-potentiating activity

Erythropoietin is the primary physiological regulator of erythropoiesis; however, in vitro studies have identified another class of mediators which appear to be important in stimulating erythroid progenitors. These factors have generally been referred to as burst-promoting activities (BPA), because they stimulate the growth of early erythroid progenitors referred to as burst-forming units-erythroid (BFU-E) which give rise to colonies of up to thousands of haemoglobinized cells. We recently reported purification of a burst-promoting activity from medium conditioned by the Mo T-lymphoblast cell line infected with human T-cell lymphotropic virus type II (HTLV-II). This purified glycoprotein of relative molecular mass (Mr) 28,000 also stimulates colony formation by more mature erythroid precursors (CFU-E) and is therefore referred to as erythroid-potentiating activity (EPA). Purified EPA specifically stimulates human and murine cells of the erythroid lineage, unlike murine interleukin-3 (IL-3) which stimulates precursor cells from all haematopoietic lineages. We report here the isolation of a complementary DNA molecular clone encoding EPA and its use in producing EPA in COS (monkey) cells and CHO (Chinese hamster ovary) cells. We also define the organization of the EPA gene in human DNA.