Effects of DNA topoisomerase II inhibitors on human bone marrow progenitor cells.

Topoisomerase II (topo II) is a target for many cytotoxic agents. Two observations, however, warrant caution in their therapeutic use: first, these agents can inhibit differentiation and second, perturbations in function render the enzyme error-prone. Illegitimate recombination events occurring at sites where topo II acts in differentiation could be particularly important in the development of secondary malignancies (relatively frequent after therapy with agents that target topo II). Topo II inhibitors are heterogeneous in mechanisms of action; in site-specificity of cleavable complex 'entrapment' (where present) and in the relative potency against the two topo II isoforms, all potentially influencing the site of maximum DNA damage. The object of this study was to examine the effect of topo II inhibitors on human haemopoietic precursor cells, to determine which have most impact on differentiation. We selected two which act via cleavable complex entrapment, but with different site preferences (m-AMSA and VP-16), and two acting via other mechanisms (merbarone and fostriecin). VP-16 and m-AMSA showed similar patterns with low dose stimulation of granulocyte-macrophage colony formation and high dose inhibition of all colony types. The stimulation was accompanied by an increase in colony size and blast content, consistent with a low dose inhibition of differentiation. Forstriecin, in contrast, stimulated predominantly mixed and erythroid colonies. Merbarone failed to increase colony formation. Neither produced substantial inhibition of colony formation. The effects on granulocyte-macrophage progenitors were confirmed using 7-day suspension cultures, using nitroblue tetrazolium (NBT) reduction and 3-4,5,dimethylthiazol 2,5-diphenyl tetrazolium bromide (MTT) assays for differentiated cells and total cell mass, respectively. These results demonstrate that the effects of topo II inhibitors on haemopoietic cell proliferation and differentiation are agent-specific and can involve lineage-restricted partial inhibition of differentiation.

Dissociation of the proliferation and differentiation stimuli of granulocyte colony-stimulating factor (G-CSF).

One proposed therapeutic application of granulocyte colony-stimulating factor (G-CSF) is in differentiation induction therapy of myelodysplastic states (MDS) or acute myeloid leukemia (AML). G-CSF however has a substantial growth including effect which limits its potential as a differentiation inducing agent. We have therefore made a systematic search for agents which might restrain the proliferative effects of G-CSF whilst retaining the differentiation stimulus. Of all the agents we have tested on human bone marrow progenitor cells: (6-thioguanine, all-trans retinoic acid, vincristine, recombinant human alpha-2b and gamma-interferon) only the latter abolished the stimulation of cell growth and retained, or possibly increased, the differentiation effect of G-CSF. The antiproliferative drugs 6-thioguanine and vincristine both antagonized the neutrophil-granulocyte differentiation inducing action of G-CSF. Retinoic acid and alpha-2b interferon both had weak effects on proliferation and failed to enhance differentiation. These results suggest that it may be possible, by combining G-CSF with a suitable second agent, to utilize its substantial differentiation inducing effect without incurring the potentially hazardous effects of increased leukemic cell growth.

Acidic isoferritin stimulates differentiation of normal granulomonocytic progenitors.

Acidic isoferritin (AIF) has been shown to be released by cells from patients with leukemia and to have an inhibitory effect on the growth of normal granulomonocytic (GM) progenitors (leukemia-inhibitory activity) during the S phase of the cell cycle. AIF is also produced by normal mature cells of the monocyte-macrophage lineage. We studied the effects of AIF on the differentiation of normal GM progenitors and found an increase in the number of mature cells in AIF-exposed cultures. This increase did not occur when AIF was pretreated with anti-heart ferritin antiserum or when basic isoferritin was used in the place of AIF. The influence of AIF was not mimicked by removing S phase cells by pretreatment with a pulse of high specific activity tritiated thymidine. Thus, the apparent differentiation-stimulating effect of AIF is not likely to be due to selective removal of immature dividing cells. The results suggest that AIF-inhibitory activity on the proliferation of GM progenitors might at least in part be mediated by a stimulus for differentiation of the target cells, thus regulating the number of mature cells which might be formed by a single progenitor cell.

Evidence for the involvement of DNA topoisomerase II in neutrophil-granulocyte differentiation.

Agents that slow cellular proliferation usually stimulate myeloid differentiation. The demonstration in this report of an anomalous inhibitory behavior of the epipodophyllotoxin VP16-213, an agent known to inhibit the enzyme DNA topoisomerase II, prompted us to investigate the role of this enzyme in both changes in DNA supercoiling and in DNA strand breakage and reunion events occurring during the induction of neutrophil-granulocyte differentiation. We recently reported that retinoic acid, an inducer of granulocytic differentiation, stimulates transient relaxation of DNA supercoiling. We now show that this is associated with the formation of small numbers of protein-linked DNA breaks (a characteristic of topoisomerase reactions). Both events are perturbed by VP16-213, and since this agent inhibits subsequent differentiation, these observations raise the possibility of a role for DNA topoisomerase II in granulocytic differentiation. The possible relevance of these findings to mechanisms of leukemogenesis is discussed.

Differentiation induction in myelodysplasia and acute myeloid leukaemia: use of synergistic drug combinations.

DNA synthesis inhibitors and vincristine greatly enhance the response of leukaemic and dysplastic cells to differentiation inducing agents such as retinoic acid (RET). Differentiation induction therapy is an attractive therapeutic approach in myelodysplasia (MDS) and in acute myeloid leukaemia (AML) in the elderly, since it should be possible to increase the production of mature cells, at the expense of precursor cells, without incurring the complications of intensive cytotoxic therapy. Single agent differentiation therapy has, however, not been highly successful. We have therefore investigated the use of synergistic combinations of agents. We treated nine patients (6 with MDS, 3 with AML) with 13-cis-retinoic acid (up to 100 mg/m2/day) in combination with either 6-thioguanine (20-40 mg/day in 14-57 day courses) or with vincristine (1-2 mg as a single injection during a four-day course of RET). Seven patients responded with an increase in the mature cells of at least one haemopoietic lineage. A concomitant decrease in marrow blasts was observed in 3/4 responding patients. The retention of dysplastic and karyotypic abnormalities and lack of a hypoplastic phase all suggested that differentiation induction was occurring in vivo. Prior failure to respond to therapy with single agents (RET in two and cytosine arabinoside in five patients) suggests that the synergy observed in vitro operates in vivo. In-vitro studies on marrow cells from seven patients demonstrated synergistic differentiation induction in 6/7 samples. The seventh patient was one of the two who did not respond clinically. The second of these clinically unresponsive patients had cells which were relatively refractory to RET in vitro, suggesting that in-vivo and in-vitro responses may be related.

Synergistic interaction between differentiation inducers and DNA synthesis inhibitors: a new approach to differentiation induction in myelodysplasia and acute myeloid leukaemia.

Numerous agents induce differentiation and maturation of neoplastic and dysplastic myeloid cells in vitro and some of these agents have been used with limited success in the treatment of patients with myelodysplastic syndromes (MDS) and myeloid leukaemias. We recently proposed that physiological and pharmacological agents which enhance differentiation and maturation in vitro act by two fundamentally different routes: (1) by hastening the progression through various differentiation/maturation steps; (2) by slowing proliferation (usually by inhibition of DNA synthesis). In order to test this thesis we looked for synergistic effects on differentiation using pairs of agents from the two groups in cultures of cells from myelodysplastic and acute myeloid leukaemia (AML) patients and from normal marrow donors. The results with three MDS, two AML and three normal samples show that combinations of differentiation inducing agents (retinoic acid, N-methylformamide) with DNA synthesis inhibitors (6-mercaptopurine, cytosine arabinoside and aphidicolin) produce a differentiation inducing effect equivalent to that of 10-100, or even 1000 fold higher concentrations of single agents. Myelotoxic effects in vitro were not synergistic. The use of these synergistic combinations should greatly enhance the usefulness of differentiation inducers in the therapy of MDS and myeloid leukaemia.

Differentiation of human acute myeloid leukaemia cells in response to exogenous and endogenous stimuli: different patterns of response suggested by a bioassay system.

The object of this study was to devise quantitative bioassay systems suitable for the analysis of differentiation in acute myeloid leukaemia (AML) in response to both endogenous (bone marrow) and exogenous stimuli. Dose response analyses of the relationship between exogenous differentiation stimulus (supplied by peripheral blood mononuclear leucocytes) and clone cell maturity in double layer semi-solid agar cultures, were used to identify a measure of response with a linear relationship to differentiation stimulus on which bioassay systems could be based. All 7 AML samples tested at presentation and 1 sample from a patient during the regenerative phase after therapy, exhibited some response to exogenous differentiation stimulus. Only in the latter was there no significant difference in the response of test and reference cells. The other 7 showed marked variation in the pattern of the response in vitro. There was no close correlation between differentiation capacity in vitro and extent of differentiation observed in vivo. This discrepancy might be related to varying availability of endogenous differentiation stimulus in different patients. The technique for assaying differentiation response was adapted to demonstrate and quantify the endogenous differentiation stimulus (the stimulus provided by the leukaemic patient's marrow cells). The results suggest that in some patients the level of this stimulus may be a major determinant in the level of differentiation achieved in vivo.

The identification of mixed granulocytic-erythrocytic colonies in vitro.

The identification of mixed granulocytic-erythrocytic colonies in culture poses special problems. First, methods based on the detection of peroxidase-like activity of red-cell hemoglobin using benzidine can give false-positive results with eosinophilic cells in colonies. Secondly, the use of the color of hemoglobin itself to identify mixed colonies prevents the detection of those colonies where the erythrocytic component is small or contains relatively little hemoglobin. We present here a new method using O-Dianisidine (O-D) to detect red-cell peroxidase activity. Subsequent staining with Luxol fast blue showed that eosinophils did not give a positive reaction with O-D. The technique also has the advantage that the whole culture gel is processed and that there is no need to pick off individual clones for identification. Using the combined stain O-D and Luxol fast blue most lineages within clones can be discriminated.

DNA strand breakage and ADP-ribosyl transferase mediated DNA ligation during stimulation of human bone marrow cells by granulocyte-macrophage colony stimulating activity.

ADP-ribosyl transferase (ADP-RT) is a chromatin-bound nuclear enzyme catalysing the transfer of ADP-ribose from NAD+ to chromatin proteins. The enzyme is activated by DNA strand breaks and has been suggested to have roles in both DNA repair (via its effect on DNA ligase II) and in differentiation. We recently demonstrated that specific inhibitors of ADP-RT preferentially inhibit differentiation of human granulocyte-macrophage progenitor cells to the macrophage lineage and that the specific proliferation/differentiation stimulus granulocyte-macrophage colony stimulating activity (GM-CSA) activates ADP-RT in human marrow cells within 3 h of exposure. The purpose of this study was to investigate the role of ADP-RT in monocyte-macrophage differentiation. By altering the time of addition of ADP-RT inhibitor it was demonstrated that maximal inhibition of macrophage differentiation only occurs when the inhibitor is added within the first 24 h of culture. This suggests that it is an early event during the induced differentiation of granulocyte-macrophage progenitor cells which requires ADP-RT. Fluorometric assay of the level of DNA strand breaks showed that GM-CSA induces DNA strand breaks which are rapidly ligated only if ADP-RT is available. These data and those of our earlier studies suggest that DNA rearrangement may be involved in differentiation of granulocyte-macrophage progenitors to the monocyte-macrophage pathway. Such a DNA rearrangement could provide a molecular basis for commitment of multipotent progenitors to a single lineage.

Role of ADP-ribosyl transferase in differentiation of human granulocyte-macrophage progenitors to the macrophage lineage.

Nuclear adenosine diphosphate-ribosyl (ADP-ribosyl) transferase is a chromatin-bound enzyme catalyzing the transfer of ADP-ribose from NAD+ to chromatin proteins. The physiologic function of this covalent modification of chromatin has not been fully established, but roles in both DNA repair and in differentiation have been proposed. We demonstrate that three specific inhibitors of ADP-ribosyl transferase (5-methylnicotinamide, 3-methoxybenzamide, 3-aminobenzamide) inhibit differentiation of human granulocyte-macrophage progenitor cells to the macrophage lineage. Differentiation to the neutrophil-granulocyte lineage is much less affected. The inhibition of macrophage differentiation seems to relate to the ability of these compounds to inhibit ADP-ribosyl transferase. A structural analogue (3-methoxybenzoic acid), which is not inhibitory for the enzyme, did not inhibit macrophage differentiation. Additional evidence for a role of ADP-ribosyl transferase in the differentiation of granulocyte-macrophage progenitors was obtained from experiments in which enzyme activity levels were measured in permeabilized marrow cells. Marrow cell ADP-ribosyl transferase activity increased after 3-hr stimulation by the differentiation/proliferation stimulus--granulocyte-macrophage colony-stimulating activity (GM-CSA). Unstimulated marrow cells showed low or undetectable levels of enzyme activity.

CFUGEMM, CFUGM, CFUMK: analysis by equilibrium density centrifugation.

A modification of the technique of Fauser and Messner was used for the culture of human multipotent hematopoietic progenitors. This modified technique shows a linear relationship between mixed granulocytic-erythrocytic colonies and the number of cells plated, even at extremely low cell doses (10(4) cells/dish) and is therefore a more suitable assay system for cell separation studies than the original non-linear method. The buoyant density of CFUGEMM (colony forming unit granulocytic-erythrocytic-megakaryocytic-macrophage) was determined using equilibrium density centrifugation. CFUGEMM were of lower buoyant density than the majority of nucleated marrow cells. Substantially enriched populations of CFUGEMM could be obtained with a single density separation procedure. The density distribution profile for CFUGEMM was also distinct from the density distribution of granulocyte-macrophage colony forming cells (CFUGM), the latter being of somewhat greater buoyant density than the former. Cells which formed clones containing only megakaryocytes in culture (CFUMK) had an intermediate density between that of CFUGEMM and CFUGM. The morphological characteristics of these progenitor cells were studied using correlation analysis. Results suggested that the CFUGEMM correspond to transitional cells without granules, the CFUGM to transitional cells with 1-4 granules and the heterogeneous group of GM-cluster forming cells to a broad category including myeloblasts, promyelocytes, myelocytes and metamyelocytes.

The production of terminal deoxynucleotidyl transferase (TdT) positive cells in cultures of human pluripotent hemopoietic progenitor cells.

A transient increase in terminal deoxynucleotidyl transferase positive (TdT+) cells was observed during the early phase of (less than or equal to day 5) cultures supporting the growth of pluripotent myeloid progenitor cells (CFU-mix). T-cell growth-promoting medium and erythropoietin were not required. The rapidity with which TdT+ cells appeared in cultures and the results of cultures where TdT+ cells were high initially (greater than 800 cells/culture) were not consistent with their having been produced by proliferation of pre-existing TdT+ cells from the bone marrow inoculum. The results suggest production of TdT+ cells from a TdT-negative precursor either by altered enzyme expression or by production of TdT+ progeny.

Use of bone-marrow culture in prediction of acute leukaemic transformation in preleukaemia.

Bone-marrow granulocyte-macrophage progenitor cell proliferation and regulatory factor (colony-stimulating activity; CSA) production were assessed at presentation and, if possible, subsequently in twenty-one patients with dysmyelopoiesis and less than 5% bone-marrow blasts. Seven patients underwent acute leukaemic transformation 1-35 months after the first marrow culture. Assay of bone-marrow endogenous CSA proved the most useful prognostic test. The rate of transformation in the seven patients with raised CSA at presentation was significantly greater (five transformed and one died at or before 4 months) than that in the fourteen patients with normal, low, or undetectable CSA (two transformed at 27 and 35 months). In one of the latter an increase in bone-marrow CSA occurred 6 weeks before transformation (serial marrow samples were not available in the other case). No other marrow culture feature measured, including the presence or absence of granulocyte-macrophage colony-forming cells and total clone numbers (colonies and smaller clusters) was as useful.

Brief report: qualitative differences in the biological activity of two sources of colony stimulating factor: GCT conditioned medium and leucocyte feeder layers.

A most stringent test for the non-identity of biologically active materials is provided by the techniques used for parallel line type bioassay of these materials. We have studied commercially available GCT conditioned medium using a parallel line type biological assay for granulocyte-macrophage colony stimulating factors (GM-CSF) and mononuclear leucocytes as a reference source of GM-CSF. Only test and reference GM-CSF sources which have active constituents with identical dose response characteristics can produce parallel line displacement in this assay system. GCT conditioned medium failed to produce parallel displacements in five different assays. We must therefore conclude that one or more active factors from the test and reference sources of GM-CSF (GCT conditioned medium and mononuclear leucocytes) are not identical. This indicates a need for caution when substituting GCT conditioned medium for leucocyte feeder layers in human bone marrow cultures.

The use of image analysis for estimation of growth in bone marrow cultures.

An image analysis system was used to assess growth in agar bone marrow cultures. The technique has advantages over visual counting techniques; it saves time, increases objectivity, reduces operator errors, and may be used to estimate total cell growth or mean clone size. Either measurement may be used to determine activity levels of factors that affect growth such as colony stimulating factors. The advantages and limitations of the technique will be discussed.

Differentiation restriction in the neutrophil-granulocyte, macrophage, eosinophil-granulocyte pathway: analysis by equilibrium density centrifugation.

Bone marrow culture techniques and equilibrium density centrifugation of human bone marrow cells were used to analyse the neutrophil-granulocyte, macrophage and eosinophil-granulocyte progenitor hierarchy. The buoyant density of progenitor cells changes as cells differentiate down the granulocyte-macrophage pathway and this allows the construction of a density 'map' of the points at which differentiation decisions are made. Unipotent progenitors, neutrophil-granulocyte (G), monocyte-macrophage (M), eosinophil-granulocyte (Eo), are more dense than bi- and tripotent progenitors (GM and EoGM) and have a lower 7-day proliferative capacity (assessed as the clone size achieved in maximally stimulated agar cultures). Experiments in which marrow cells were separated on a basis of their density and either cultured in agar immediately or after an interval of 6 days in suspension culture, were performed to establish the density of the cells which give rise to each type of progenitor, i.e. to investigate parent-progeny relationships. In each case the parent cells were of lower density than the unipotent or bipotent progenitor in question. The ability to separate, at least partially, unipotent, bipotent and multipotent cells of closely related lineages is important since it facilitates studies of the intracellular events taking place as restriction of the cell's differentiation options takes place.

Relative and absolute deficiency of bone marrow endogenous colony stimulating activity in acute myeloid leukemia: relation to response to chemotherapy.

The purpose of this study was to determine possible mechanisms for the recently observed association between insensitivity of acute myeloid leukemia (AML) clonogenic cells to colony stimulating activity (CSA) and poor response to induction chemotherapy. The bone marrow endogenous CSA was determined using semi-solid agar cultures by measuring the response of the AML patients' own clonogenic cells to endogenous CSA. The results show that whereas 31% (5/16) of patients at presentation have deficient bone marrow endogenous CSA production, over 50% (11/21) have relative deficiency of endogenous CSA, due to insensitivity of the patients' clonogenic cells to CSA. Although there is an association between relative deficiency of endogenous CSA and a poor response to therapy, the relationship is not close enough to explain the previously observed highly significant correlation between insensitivity to CSA and poor response to therapy. The CSA-insensitive phenotype and poor response to therapy, one via the tendency to relative endogenous CSA deficiency in the CSA-insensitive group and another via some additional feature of these poor response AML phenotypes which is independent of the presence or absence of endogenous CSA deficiency.

Distinct T lymphocyte subsets affect granulo-monocytic differentiation and proliferation.

Bone marrow culture techniques using semi-solid support-media provide an opportunity to observe granulocyte-macrophage progenitor cells proliferating and differentiating in vitro. Cells from the bone marrow and peripheral blood produce specific growth factors which stimulate both the proliferation and differentiation processes (collectively termed colony-stimulating factors or activity, CSF/CSA). Even highly purified CSA induces proliferation and concomitant differentiation suggesting that the two processes are inseparable. We here present evidence to suggest that the two processes are, at least in part, individually regulated. We observed delayed differentiation by morphological, cytochemical and functional criteria in granulocyte-macrophage clones formed in cultures in which the growth stimulus was supplied by marrow or blood cells which have been depleted of T lymphocytes bearing the OKT3/MBG6 antigen(s) by complement lysis. The proliferation stimulus was unaffected. T cell depletion using another monoclonal antibody, OKT11a (which removed an overlapping but not identical population of T cells), did not produce the same effect but did increase the level of CSA produced by the remaining marrow cells. Selective replacement experiments using OKT4+ or OKT8+ cell preparations suggested that removal of an MBG6+, OKT3+, OKT8+, OKT4-lymphocyte subset is responsible for the effect on differentiation.