J. J. Berney

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.

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.

Stimulation of Human Haemopoietic Cells by Colony Stimulating Factors: Sensitivity of Leukaemic Cells

Summary. The sensitivity of populations of human granulocyte precursors to factors with colony stimulating activity (CSA) was assessed in agar culture in vitro. The mean threshold of stimulation was estimated by analysis of dose‐response curves of clone growth against concentration of CSA. This method of assessment has the advantage that CSA production by cells contaminating the population under test does not affect results. Marrow cells from patients with acute myeloid leukaemia were less sensitive to CSA than marrow cells from normal individuals. Sensitivities of cells from chronic granulocytic leukaemia and from chronic myelomonocytic leukaemia were within the normal range, but this group also tended to require more CSA than controls. In addition, sensitivities of granulocyte precursors from patients with acute myeloid leukaemia were closely related to the culture pattern, and thus to the remission probability. The significance of this relationship is discussed.

Stimulation of Human Haemopoietic Cells by Colony Stimulating Factors: Adherent Cell Dependent Colony Stimulating Activity in Human Serum

The number of granulocyte‐macrophage clones formed in agar culture of bone marrow is dependent on levels of colony stimulating activity (CSA) a proposed in vivo haemopoietic regulator. A dose‐response relationship for stimulation of human haemopoietic cells by CSA is demonstrated, which could be explained by thresholds of stimulation to cell division following a normal distribution. A simple method for the comparison of activities of test and control sources of CSA is presented. The apparent potentiating effect of the addition of two sources of CSA is explained by this dose‐response relationship. Haemopoietic cells from patients with chronic granulocytic or acute myeloid leukaemia showed the same dose‐response relationship. CSA levels in normal human sera were greatly reduced by assay in the absence of adequate numbers of bone marrow ‘adherent cells’(cells adherent to nylon or plastic) or peripheral blood leucocytes, suggesting the presence of two kinds of CSA in human serum, one dependent on the presence of bone marrow adherent cells and one effective in their absence. Reduction of numbers of nonspecific esterase positive cells in the bone marrow sample correlated with reduction in the stimulating effect of serum. In all sera tested, adherent cell dependent CSA was the major component.

Proliferative capacity, sensitivity to colony stimulating activity and buoyant density: linked properties of granulocyte-macrophage progenitors from normal human bone marrow.

Abstract Phenotypic differences between acute myeloid leukaemia (AML) cells and normal cells can only be attributed to abnormal AML cell properties if comparisons have been made with the precise normal counterpart cell. When AML clonogenic cells are compared with the normal bone marrow cells which form clones of granulocytes and macrophages in agar culture, differences are detected. These include lower average buoyant density and varying degrees of reduced sensitivity to the specific regulator granulocyte-macrophage colony stimulating activity (CSA). The degree of reduction in sensitivity to CSA is inversely related to the proliferative capacity of the individuals AML cells. This association suggests that the phenotypes of different AML cell populations might be a parody of the phenotypic heterogeneity of the normal granulocyte-monocyte progenitor population; heterogeneity which possibly reflects the maturation hierarchy of the normal population. The aim of this study was to test this thesis. The results show that buoyant density, proliferative capacity and sensitivity to CSA are heterogenous properties of normal granulocyte-monocyte progenitor cells which are linked in an orderly manner. Buoyant density and CSA sensitivity differences between AML cells and normal progenitor cells may thus indicate shifts in the location of the majority of progenitor cells on the granulocyte-monocyte pathway in AML rather than abnormal cell properties.

The Effect of Androstanes on Granulopoiesis in Vitro and in Vivo

Summary. Bone marrow culture in a patient with aplastic anaemia responding to anabolic steroid (methandienone) therapy, showed an unusually high degree of growth in unstimulated cultures. Growth in unstimulated cultures is due to factors with colony stimulating activity (CSA) released by monocyte macrophages in the bone marrow sample. These cells, which can be identified by staining for non‐specific esterase activity, were not increased in this patients marrow, implying either increased production of colony stimulating factors or increased sensitivity to these factors. Addition of methandienone or testosterone to feeder‐layers containing normal peripheral blood leucocytes increased their stimulatory activity. Addition of these drugs to feeder‐layers of CSF‐containing conditioned medium did not have this effect, implying that androstanes cause increased production of colony stimulating factors rather than increased sensitivity to them. Lack of response to androstane therapy may be related to lack of response by bone marrow monocyte macrophages or to the inability of granulopoietic cells to respond to the increased CSF production induced by androstane therapy. A means of predicting lack of response is proposed.

Sensitivity of Acute Myeloid Leukaemia Cells to Colony Stimulating Activity: Relation to Response to Chemotherapy

Summary. Agar cultures of acute myeloid leukaemia (AML) cells were used to determine the sensitivity of AML clone forming cells to the specific regulator granulocyte‐macrophage colony stimulating activity (CSA). The mean CSA threshold (amount of CSA required to stimulate 0.5 of the clone forming cells) of 27 different AML cell samples varied from normal to an approximately nine‐fold increase in CSA requirement. The degree of sensitivity to CSA at presentation was found to be closely correlated to the response to induction chemotherapy in 15 patients. There was a highly significant correlation (P<0.001) between the mean CSA threshold and the number of courses of chemotherapy required to induce remission. Also, an increased mean CSA threshold at presentation was associated with a significantly slower decline of marrow blasts following the first course of chemotherapy and a reduced remission rate. Sensitivity to CSA was closely related to the clone size achieved in maximally stimulated cultures of AML cells and this in turn also related to the remission rate and ease with which remission was achieved (presentation marrows, 39 patients). Possible reasons for the linking of these two functional properties of AML cells and their relation to response to chemotherapy will be discussed.

Divergent sensitivities of leukaemic cells to human placental conditioned medium and leucocyte feeder layers

Abstract It has been suggested that placental conditioned medium provides an alternative to leucocyte feeder layers as a source of granulocyte/macrophage colony stimulating activity for the culture of human bone marrow. The present study demonstrates important differences in the response of acute myeloid leukaemia cells to stimulation by these two sources of activity which are not revealed by tests using cells from normal individuals or patients with chronic granulocytic leukaemia. Human placental conditioned medium is not therefore a suitable substitute for leucocyte feeder layers in acute myeloid leukaemia (AML). The results also show that there are functionally distinct clone forming cell types in AML associated with different clone size potentials and response patterns.

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.

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 patients 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.