Transfection of murine multi-potent haemopoietic stem cells with an E. coli DNA alkyltransferase gene confers resistance to the toxic effects of alkylating agents.

O6-alkylguanine-DNA-alkyltransferase (ATase)-deficient murine haemopoietic stem cells were transfected, following electroporation, with a G418-selectable expression vector containing the protein coding region of the Escherichia coli ATase gene ada. Clones of cells that were resistant to G418 or the chloroethylating agent mitozolomide (Mz) were selected and most were shown to express very high levels of bacterial gene-encoded ATase. In comparison with control cells that were transfected with the parent vector, the ATase-expressing clones were considerably more resistant to the toxic effects of the methylating agents N-methyl-N-nitrosourea and methylmethanesulphonate or the chloroethylating agents Mz or taurine chloroethylnitrosourea, but unchanged in their susceptibility to the bis-chloroethylating agent nitrogen mustard.

Phase I/II study of recombinant human granulocyte colony-stimulating factor in patients receiving intensive chemotherapy for small cell lung cancer.

Twelve patients with advanced small cell carcinoma of the bronchus were treated by continuous infusion of recombinant human granulocyte colony-stimulating factor (rhG-CSF) at the following dose levels: 1 microgram, 5 micrograms, 10 micrograms, 20 micrograms and 40 micrograms kg-1 day-1 for 5 days. No toxicities resulted from the treatment and in all 12 patients the number of peripheral neutrophils increased rapidly to a maximum of 100 x 10(9) l-1 at 10 micrograms kg-1 day-1. The neutrophils were shown to be functionally normal in tests of their mobility and bactericidal activity.

Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells.

When highly enriched multipotential spleen colony forming cells (CFU-S) obtained following fluorescence activated cell sorting (FACS-CFU-S) are cultured on marrow stromal cells, they undergo proliferation and development to produce mature haemopoietic cells (Spooncer et al., Nature, 316:62-64, 1985). We now show that FACS-CFU-S behave in a similar way when cultured on monolayers of 3T3 cells, indicating that the 3T3 cells can supply at least part of the environment which is representative of marrow stromal cells and provide, therefore, a system for studying stromal cell: haemopoietic cell interactions.

Developmentally-related changes in surface membrane glycopeptides of murine haemopoietic cells.

We have carried out a comparative study of mature murine granulocytes with two immature haemopoietic cell lines (multipotential cells, FDCP-Mix, and granulocyte progenitor cells, FDCP-2) with respect to the structure and composition of their surface membrane glycopeptides. The glycopeptides were labelled biosynthetically by incubation of the cells for 1-3 days with [3H]glucosamine. Cell-associated glycopeptides were released by treatment with trypsin and the trypsin extract was exhaustively digested with Pronase to remove most residual peptide.

Growth and differentiation in the hemopoietic system.

Hemopoiesis is regulated by a complex series of interactions, including interactions among hemopoietic cells themselves, hemopoietic cells and the extracellular matrix, hemopoietic cells and marrow stromal cells, and hemopoietic cells and growth factors. In vitro culture systems have allowed a reductionist approach to the solution of these various problems and have facilitated experiments at the mechanistic level. The hemopoietic system is organized hierarchically with multipotential self-renewing stem cells, committed progenitor cells, and mature cells.

Differentiation apparently repressed by the nucleus. Rapidly-induced pigmentation of enucleated melanoma cells.

There is evidence for cytoplasmic control over gene expression in cell differentiation, but still very little is known of the intracellular mechanism, nuclear, cytoplasmic, or both, which actively initiates the differentiation of one cell type into another. Here the role of the cytoplasm was examined in the induction of differentiation of cultured mouse melanoma cells by melanocyte-stimulating hormone and alkaline medium. Intact cells were compared with cytoplasts, cells enucleated by centrifugation in the presence of cytochalasin D (CD).

Phorbol esters activate protein kinase C and glucose transport and can replace the requirement for growth factor in interleukin-3-dependent multipotent stem cells.

Interleukin 3 (IL-3) promotes the survival, proliferation and development of progenitor cells from several distinct haemopoietic lineages and can also stimulate the self-renewal of stem cells. We have explored the mode of action of this growth factor in promoting survival and proliferation, using a multipotent haemopoietic stem cell line FDC-Mix 1. In the absence of IL-3 these cells died within 16-48 h.

Increased experimental metastatic capacity of a murine melanoma following induction of differentiation.

Because of the interest in possible links between defective differentiation and cellular malignancy, the effects were examined of induced cell differentiation upon the experimental metastatic potential of the sublines F1 and F10 of the B16 mouse melanoma. These cell lines normally have low and high rates, respectively, of colonization of the lungs of mice after i.v.

The myelosuppressive effect of recombinant interferon gamma in short-term and long-term marrow cultures.

The effect of a highly purified human gamma IFN (r-gamma-IFN) on the growth of haemopoietic progenitors was examined in soft agar assays and in long-term bone marrow cultures. r-gamma-IFN reduced colony formation by progenitor cells of the granulocyte/macrophage lineage (GM-CFC), the erythroid lineage (BFU-E) and multipotent cells (GEMM-CFC), and suppressed haemopoiesis in long-term culture in a dose-related fashion. At high doses (1000 units/ml) r-gamma-IFN appeared to be toxic to the stromal cells of the bone marrow.

Growth and development in the haemopoietic system: the role of lymphokines and their possible therapeutic potential in disease and malignancy.

Some cells can produce soluble factors which modify the behaviour and/or growth pattern of other cells. These 'factors' are broadly referred to as cytokines and may be further classified on the basis of the cells which were first characterized as the producer cells. For example, lymphokines are the soluble products of lymphocytes and monokines are the soluble factors released by mononuclear phagocytes.

Comparison of haemopoiesis in young and old mice.

Haemopoietic status and functions have been compared in young (2-3-month-old) and old (2-2.5-year-old) BDF1 mice. The parameters measured include total marrow cellularity, CFU-S, CFU-mix, GM-CFC, BFU-E and CFU-F.

Perturbed hemopoiesis and the generation of multipotential stem cell clones in src-infected bone marrow cultures is an indirect or transient effect of the oncogene.

Multipotential stem cell lines, derived specifically from long-term bone marrow cultures infected with a recombinant retrovirus carrying v-src, lack v-src. Stable consequences thus result from transient actions or indirect effects of v-src on other cells, with the latter possibility being favored by its mosaic expression in marrow cultures.

Reconstitution of haemopoietic system with autologous marrow taken during relapse of acute myeloblastic leukaemia and grown in long-term culture.

In long-term cultures of bone marrow from a patient with acute myeloblastic leukaemia (AML) the leukaemic chromosome marker, which was present in a proportion of the marrow cells even during morphological remission, was not detectable after seven days of culture. After florid relapse of AML, large-scale bone marrow cultures were established while the patient received conditioning with cyclophosphamide and total body irradiation as for an allogeneic bone marrow transplant. Marrow cultured for ten days was reinfused and three and six months later the patient was in clinical remission with a normal karyotype.

Altered stem cell (CFU-S) function following infection of hematopoietic cells with a virus carrying V-src.

Long-term murine bone marrow cultures were used to support the growth and development of hematopoietic cells. After hematopoiesis was established, the cultures were infected with a recombinant murine amphotropic virus carrying the avian sarcoma virus src gene and the CFU-S kinetics were examined. The CFU-S from the src-infected cultures displayed a reduced seeding efficiency in the standard spleen colony assay.

Combined use of lectin affinity chromatography and endo-beta-galactosidase to study polylactosamine sequences isolated from haemopoietic cell surfaces.

The trypsin-sensitive glycopeptides from cell surfaces of a multipotential murine haemopoietic cell line (DE) have been studied using serial lectin affinity chromatography on columns of immobilized lentil lectin (LCA), concanavalin A (Con A), and wheat-germ agglutinin (WGA). WGA-binding material consisted of glycopeptides that failed to bind to LCA and Con A. Step elution from the WGA-column with 0.

Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors.

Interleukin-3 (IL-3)-dependent cell lines (FDCP-mix) were cloned and isolated from long-term bone-marrow cultures infected with src-MoMuLV. These cell lines have many of the characteristics of hematopoietic stem cells. Early isolates of the FDCP-mix cells form spleen colonies in irradiated mice and establish long-term hematopoiesis on irradiated marrow stroma in vitro in the absence of IL-3.

The kinetic response of haemopoietic precursor cells, in vivo, to highly purified, recombinant interleukin-3.

Interleukin-3 is a murine haemopoietic cell growth factor which has now been prepared in recombinant form, rIL-3. This purified material has been shown to act, in vitro, in a comparable manner to the native material and has recently been shown to have an in vivo effect on the committed, in vitro colony-forming cells. We have examined the effects, in vivo, of low acute and chronic infusion doses on the pluripotent haemopoietic spleen colony-forming cells, CFU-S, (stem cells).

Characterisation of monoclonal antibodies which specifically recognise the human erythrocyte glucose transport protein.

Two monoclonal antibodies (mabs) of subclass IgG1 have been raised against the human erythrocyte glucose transport protein. The mabs bound to the purified glucose transporter in both its membrane-bound and detergent-solubilised forms. However, they exhibited little or no binding to the detergent-solubilised nucleoside transport protein, which is present as a minor contaminant in the glucose transport protein preparation.

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.

Defective ability to self-renew in vitro of highly purified primitive haematopoietic cells.

Stromal cells play a critical role in haematopoiesis, both in a permissive and, probably, in a directive manner. Study of the interactions between stromal cells and haematopoietic stem cells, however, is difficult to perform using whole bone marrow, in which stem cells are indistinguishable from precursor cells and maturing haematopoietic cells, and where stromal and haematopoietic cells co-exist in a heterogeneous mixture. We have purified primitive haematopoietic spleen colony-forming cells (CFU-S) using fluorescence-activated cell sorting (FACS) and produced CFU-S populations which approach 100% purity (ref.

Inhibitors of cholera toxin-induced adenosine diphosphate ribosylation of membrane-associated proteins block stem cell differentiation.

Two potent inhibitors of mono-adenosine diphosphate (ADP) ribosylation have recently been described and characterized, named p-methoxylbenzylaminodecamethylene guanidine sulfate (MBAMG) and benzylaminododecylguanine hydrochloride (BADGH). We have used these agents to investigate the role of ADP ribosylation in hematopoiesis using long-term marrow cultures. The addition of MBAMG (10(-6) mol/L) or BADGH (5 X 10(-4) mol/L) led to both an inhibition of mature cell production and the development of colony-stimulating factor (CSF-1)-responsive GM-CFC, but had no effect upon spleen colony-forming units (CFU-S) or on progenitor cells which respond to the multilineage stimulating factor present in WEHI-3B cell-conditioned medium.