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In this study we explored the effects of bryostatin-5 on the clonogenic response of normal bone marrow mononuclear (BM) cells and HL60 myeloid leukemia cells. Leukemic HL60 colony formation was strongly inhibited by bryostatin-5 depending on dose and schedule. An inhibitory effect on HL60 colony formation was readily demonstrated after 1 h of exposure, reaching a maximal inhibitory effect at 96 h. Normal BM cells differed in their clonogenic response: short-term exposure to bryostatin-5 resulted in increased clonogenicity while longstanding exposure to bryostatin-5 permitted the survival of a substantial fraction of committed progenitors. This differential modulation of normal and leukemic myeloid clonogenicity by bryostatin-5 suggests a possible role for bryostatin-5 in the treatment of acute myeloid leukemia.

In order to study the adhesive interactions of the human bone marrow microenvironment and acute myeloid leukaemic cells, we investigated the binding capacity of KG-1 cells upon human long-term bone marrow cultures derived from 17 healthy volunteers and 12 patients with acute myeloid leukemia. Adhesion was measured using a 51-chromium labelling assay. Adhesion of KG-1 cells upon 'normal' stromal layers: 33% +/- 4.0, n = 17 (mean +/- SEM) was higher as compared to the binding to 'leukaemic' stromas: 24% +/- 3.7, n = 12 (p < 0.05). Blocking monoclonal antibodies against adhesion molecules reduced the binding of KG-1 cells upon 'normal' stroma, when anti-VLA4 (p < 0.03), anti-Mac1 (p < 0.03) and anti-p150/95 (p < 0.04) were used. Binding of KG-1 cells on 'leukaemic' stromas was partly inhibited by anti-VCAM1 (p < 0.03). Blocking achieved by single or combined antibodies was never complete, suggesting that the adhesion is a multifactorial process, including a variety of adhesion molecules and/or adhesion mechanisms.

1.25 (OH)2D3 is a potent inducer of differentiation of leukaemic cells into a monocytic direction. However, therapeutic application is difficult because of the development of hypercalcaemia. We examined a novel vitamin D analogue, MC 903, which is at least 100 times less effective on calcium metabolism in rats than 1.25 (OH)2D3. Using the HL-60 cell line, differentiation was measured with a comprehensive panel of qualitative and quantitative parameters. Development of monocytic cells was shown morphologically, immunophenotypically and functionally by increased capability of reducing NBT (vs cultures without MC 903, p less than 0.0001) and by qualitatively and quantitatively increased non-specific esterase activity. Furthermore, a concomitant decreased activity of myeloperoxidase and lactate dehydrogenase was noticed. In conclusion, MC 903 is a potent inducer of monocytic differentiation, comparable with 1.25 (OH)2D3 and will therefore be an interesting and potential therapeutic agent for studies in human acute leukaemia.

Acute myeloid leukaemia (AML) cells have a variable capacity to egress from bone marrow into peripheral blood. This may be due to a variable lack of adhesion molecules on leukaemic cells. The expression of VLA1, 3, 4, 5, 6, beta 1-chain, LFA1, beta 2-chain, ICAM1 and NCAM appeared to be higher in bone marrow as compared to peripheral blood leukaemic cells, although this only reached significance for beta 1-chain (p less than 0.01). The number of cases with more than 20% positive cells in bone marrow leukaemic cells was lower in immature FAB-subtypes (M1, M5a) as opposed to more mature subtypes (M2, M3, M4, M5b) for the adhesion molecules tested. This reached significance for VLA5 (p less than 0.05) and beta 1-chain (p less than 0.007), while there was trend for VLA4. It is discussed that VLA4 and 5 may play a role in the release of leukaemic cells from the bone marrow.

Freshly obtained leukemic cells from 64 patients with acute myeloid leukemia were investigated. Intracellular protein and enzyme content (myeloperoxidase (MPO), acid esterase with alpha naphthyl acetate (ANAE) and alpha naphthyl butyrate (ANBE) and lactate dehydrogenase) were measured together with the cell diameter and compared with FAB-classification, cytochemical staining and clinical features. Mean values of MPO and acid esterase discriminate between the pure myeloid leukemias, although overlap between various subgroups is considerable. No correlation between cytochemical and quantitative enzyme determination was found. The logistic regression analysis revealed a dependency of the occurrence of complete remission on protein and ANBE (p less than 0.01) content. We concluded that quantitative enzyme determination is an easily measurable parameter of maturation and may have prognostic significance.

Bone marrow stromal cells are critical for the proliferation and differentiation of hemopoietic stem cells. The hemopoietic microenvironment is reproduced in long term bone marrow culture (LTBMC). Normal LTBMC versus leukemic LTBMC and their stroma conditioned medium were compared with respect to their proliferative and differentiation-inducing capacities. Myeloid leukemic cells (HL60) were layered onto LTBMCs derived from normal volunteers and patients with AML. Differentiation was measured with a comprehensive panel of maturation parameters, i.e. morphology, cytochemistry, quantitative enzyme determination, NBT test and immunophenotyping. Inhibition of proliferation occurred in all cocultures. Clear maturation in monocytic direction was obvious in one culture of HL60 cells layered onto a leukemic stroma. As stroma-derived conditioned medium has no effect, a cellular interaction seems involved. These observations support not only the concept that normal stroma influences leukemic cell growth but also that leukemic stroma can modulate cell growth and maturation.

Introduction of the maturation index (MI) as a measure for the degree of maturation improved the subtyping of acute myeloid leukemia (AML). A comparison is made here between the MI and the results of surface marker analysis with a panel of monoclonal antibodies (McAb) in the immunofluorescence technique. The McAb applied in 46 AML patients (greater than or equal to 15 years) were granulocyte specific (MI/N1, UJ 308, B4-3, B13-9), granulocyte-monocyte specific (OKM-1, B2-12) or had specificity for the Ia-like antigen (OKI-1), 'T-cells' (3A1), immature cells (OKT-10) or platelets (C17-28). In 32 of these patients more McAb could be investigated with specificities for granulocytes (VIM-D5), granulocytes-monocytes (RUPI-5), monocytes (MONO BRL, RUPI-4), erythrocytes (VIE-G4) and AML cells (VIM-S8). An increase in surface marker expression evident from the reaction with a number of McAb (UJ 308, B2-12, OKM-1 and OKI-1) paralleled the rise of the MI in FAB M5. A decrease of the expression of antigen detected by OKI-1 paralleled the rise of the MI in FAB M1-3. The granulocyte or monocyte specific McAb, as they are determined on normal human peripheral blood cells, did not distinguish between FAB M1-3 and M5. The maturation index seems to be a valuable tool in understanding the results of surface marker analysis.

From ten consecutive patients with acute myeloid leukemia leukemic cells were isolated and cultured with and without 10(-6), 10(-7) and 10(-8) M 1.25(OH)2D3 (vit D3), retinoic acid (RA) cytosine-arabinoside (ARA-C) and 1.0, 1.25 and 1.5% dimethyl sulfoxide (DMSO). Maturation was measured with a comprehensive panel of qualitative and quantitative parameters of maturation. Six of those ten leukemias showed significant (p less than 0.01) changes in at least three parameters after exposure to either one of the differentiation inducers. Vit D3 induced maturation in four leukemias, in three of them clearly in monocytic direction. ARA-C showed changes in one leukemia in only three parameters not pointing to either granulocytic or monocytic direction. Maturation in granulocytic direction was observed after exposure to RA in one leukemia. Maturation induction was observed in six out of ten freshly isolated leukemic cells with vit D3 being the most potent inducer of maturation in monocytic direction. The data about inducibility of maturation in freshly isolated human leukemic cells are reviewed and discussed.