Familial platelet disorder with predisposition to acute myelogenous leukaemia (FPD/AML, MIM 601399) is an autosomal dominant disorder characterized by qualitative and quantitative platelet defects, and propensity to develop acute myelogenous leukaemia (AML). Informative recombination events in 6 FPD/AML pedigrees with evidence of linkage to markers on chromosome 21q identified an 880-kb interval containing the disease gene. Mutational analysis of regional candidate genes showed nonsense mutations or intragenic deletion of one allele of the haematopoietic transcription factor CBFA2 (formerly AML1) that co-segregated with the disease in four FPD/AML pedigrees. We identified heterozygous CBFA2 missense mutations that co-segregated with the disease in the remaining two FPD/AML pedigrees at phylogenetically conserved amino acids R166 and R201, respectively. Analysis of bone marrow or peripheral blood cells from affected FPD/AML individuals showed a decrement in megakaryocyte colony formation, demonstrating that CBFA2 dosage affects megakaryopoiesis. Our findings support a model for FPD/AML in which haploinsufficiency of CBFA2 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia.

Efficient retroviral gene transfer into hematopoietic stem and progenitor cells can be achieved by co-localizing retrovirus and target cells on specific adhesion domains of recombinant fibronectin (FN) fragments. In this paper, we further optimize this technology for human CD34+ cells. Investigating the role of cytokine prestimulation in retrovirus-mediated gene transfer on plates coated with the recombinant FN CH-296 revealed that prestimulation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) CD34+ cells was essential to achieve efficient gene transfer into clonogenic cells. The highest gene transfer occurred by prestimulating PB CD34+ cells for 40 hr with a combination of stem cell factor (SCF), G-CSF, and megakaryocyte growth and development factor (MGDF) prior to retroviral infection on CH-296. Surprisingly, a prolonged simultaneous exposure of primary CD34+ PB cells to retrovirus and cytokines in the presence of CH-296 lowered the gene transfer efficiency. Gene transfer into cytokine prestimulated CD34+ bone marrow (BM) cells was not influenced by increasing the coating concentrations of a recombinant FN fragment, CH-296, nor was it adversely influenced by increasing the number of CD34+ target cells, suggesting that the amount of retroviral particles present in the supernatant was not a limiting factor for transduction of CD34+ BM cells on CH-296-coated plates. The polycation Polybrene was not required for efficient transduction of hematopoietic cells in the presence of CH-296. Furthermore, we demonstrated that repeated exposure of CH-296 to retrovirus containing supernatant, called preloading, can be employed to concentrate the amount of retroviral particles bound to CH-296. These findings establish a simple and short clinically applicable transduction protocol that targets up to 68% of BM or G-CSF-mobilized PB CD34+ cells and is capable of genetically modifying up to 17% of CD34+CD38-/dim PB cells.

Patients with certain genetic disorders can be cured by bone marrow transplantation. However, as prospective donors do not exist for most patients with potentially curable genetic abnormalities, an alternative treatment for such patients involves the transfer of cloned genes into the patient's haematopoietic stem cells followed by re-infusion of the treated cells. Retroviral vectors provide an efficient means for transferring genes into mammalian cells and have been used to transfer genes into mouse haematopoietic cells. We have now produced amphotropic retroviral vectors containing either the bacterial gene for neomycin resistance or a mutant dihydrofolate reductase gene that confers resistance to methotrexate and have used these vectors to infect and confer drug resistance to human haematopoietic progenitor cells in vitro. Transfer could be demonstrated in the absence of helper virus by using an amphotropic retrovirus packaging cell line, PA12 (ref. 9). These studies are an important step towards the eventual application of retrovirus-mediated gene transfer to human gene therapy and for molecular approaches to the study of human haematopoiesis.

Amoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F-actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F-actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross-linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin-binding proteins that are present in the cell cortex.

To determine whether leukocytes express the angiotensinogen gene, we subjected circulating rat leukocytes and murine bone marrow cells to Northern blot analysis and hybridization with homologous angiotensinogen complementary DNA. Angiotensinogen messenger RNA sequences were detected in circulating adult rat leukocytes, in murine-irradiated and nonirradiated bone marrow stromal cells, and in an adherent stromal cell line (preadipocyte). Western blot analysis of rat leukocyte homogenate showed that rat leukocytes contain two main angiotensinogen isoforms with approximate molecular weights of 46.5 and 53.9 kd. Synthesis and release of angiotensinogen protein by rat leukocytes was confirmed by immunoprecipitation of radiolabeled angiotensinogen from cell lysate and media of rat leukocytes that were metabolically labeled with M S-L-methionine. In addition, the angiotensinogen protein present in media of rat leukocytes was enzymatically cleaved by hog renin, resulting in generation of angiotensin I (305±47 pg angiotensin I per milliliter of media per hour). We conclude that circulating rat leukocytes express the angiotensinogen gene and synthesize and release angiotensinogen with the capability to generate angiotensin. Expression of angiotensinogen by leukocytes may provide a mobile angiotensingenerating system of potential importance in the regulation of local inflammatory responses, tissue injury (i.e., myocardial infarction), and arterial hypertension.

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PtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows.

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