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The hematopoietic growth factors granulocyte/macrophage colony-stimulating factor (GM-CSF) and G-CSF, available as recombinant products, stimulate the growth in culture of blasts from patients with acute myeloblastic leukemia (AML). We used cDNA probes for each gene to study the genomic organization in blast cells of 22 patients and expression in the blast cells of 18 patients. Alteration in the structure of G-CSF (two instances) and GM-CSF (two instances) was found. In two patients in whom it was possible to study DNA from bone marrow obtained at remission, the new bands detected in the leukemic cells were not found. Fifteen of 18 patients showed no RNA expression of either growth factor. Both patients with GM-CSF abnormalities as seen by Southern analysis expressed an abnormally large GM-CSF message but no G-CSF messages. One patient with an abnormal Southern pattern with G-CSF expressed normal-sized G-CSF and GM-CSF messages. The biologic significance of these findings remains to be determined. Nonetheless, the abnormal Southern patterns may prove to be useful clonal markers in the study of AML.

We have proposed that the blasts in acute myeloblastic leukemia (AML) are renewal populations maintained by a small subpopulation of stem cells. The balance between self-renewal and differentiation in blast stem cells may be an important attribute contributing to treatment outcome. Cytosine arabinoside (ara-C) is included in most chemotherapeutic regimens for the treatment of AML. When ara-C survival curves are constructed, the drug appears to be more toxic when an assay is used that detects principally self-renewing divisions, compared with a procedure that depends on terminal divisions. AML blasts usually respond in culture to myelopoietic growth factors; their response often includes a change in self-renewal, differentiation, or both. These features of the model for AML blasts led to the prediction that growth factors would alter ara-C survival curves in a way that depended on the effects of the culture conditions on self-renewal and differentiation. Four AML blast populations were chosen to test this prediction on the basis of our ability to manipulate them by adding or withholding one or more growth factors. Highly significant changes were seen in the ara-C survival curves, depending on the growth factors present in the cultures as was predicted by the observed effects of the factors on renewal and differentiation.

The blast cells of acute myeloblastic leukemia may be considered as a renewal population maintained by stem cells that are capable of both self-renewal and differentiation. Blast stem cells grow in culture usually when stimulated by growth factors normally active on myelopoietic cells. Two culture methods permit an evaluation of the balance between self-renewal and differentiation; previous studies have shown that this balance can be affected by recombinant growth factors. These include interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF), active on early cells in normal myelopoiesis, and G-CSF and CSF-1, restricted in normal hemopoiesis to the granulopoietic and macrophage/monocytic lineages, respectively. In this paper we report the results of evaluating the effects on these recombinant growth factors alone or in mixtures of two at optimal concentrations. The results were obtained either using titrations of colony formation in methylcellulose or growth in suspension. Star diagrams, a technique from exploratory data analysis, were used to provide quantitative and graphic displays of the results of the recombinant factors on the balance between blast self-renewal and differentiation. Blasts from 4 acute myeloblastic leukemia patients and one patient with the blast crisis of chronic myeloblastic leukemia were examined in detail. The great patient-to-patient variation usually observed was seen in both plating efficiency in methylcellulose and growth pattern in suspension. In spite of this variation, a common pattern of response to growth factors emerged. When the early acting factors, IL-3 and GM-CSF, were combined, the effect was quantitatively and qualitatively similar to the largest stimulation seen with either of the factors alone. In contrast, late-acting factors, G-CSF and CSF-1, influenced each other's effects when present together and each affected the activities of GM-CSF and IL-3. Notably, CSF-1, which often led to the accumulation of adherent, terminal cells in suspension, usually maintained or increased this differentiation-like activity in combination. G-CSF also favored differentiation in combination, although the effect was usually to increase the number of colonies in methylcellulose, most of which consist of blast cells incapable of further divisions. The results are discussed as they relate to the postulated structure of the blast population and the normal targets of the recombinant growth factors.

Myelopoietic growth factors are known to influence the growth in culture of malignant blast cells from human Acute Myeloblastic Leukemia (AML). We have used cDNA clones for the factor CSF-1 and its receptor fms to study DNA and RNA from the blasts of 25 AML patients. The CSF-1 gene was always in the germline configuration. CSF-1 mRNA was found in about half the blast populations. The cells were also studied for their growth properties in culture. A highly significant association was found between CSF-1 expression and poor growth in suspension culture. Most blast populations expressed fms; the number of fms expression negative samples was to small to permit the detection of any association between fms expression and growth or any interaction between the effects of the expression of the growth factor and its receptor. We propose that CSF-1 may be an important part of the mechanism determining the balance between self-renewal and determination in AML blast clones.

Granulocyte/macrophage-colony-stimulating factor (GM-CSF) is an effective growth factor for the blasts of acute myeloblastic leukemia (AML). Radioiodinated Chinese hamster ovary (CHO)-cell derived GM-CSF was prepared using Bolton-Hunter reagent to label free amino groups on the protein. Normal human neutrophils and the blast cells from AML patients were examined for binding. We found that there were fewer receptors of higher affinity on blast cells compared with neutrophils. After brief culture in suspension, receptor number increased and affinity decreased. Experiments provided evidence that GM-CSF from Escherichia coli had a higher affinity for neutrophils (kd = 20 pM) than the CHO-cell derived protein (kd = 500 pM-1 nM). This difference was reflected in the increased effectiveness of the E. coli protein over the CHO protein to stimulate colony formation in both normal bone marrow cells and AML blasts.

Recombinant hemopoietic colony-stimulating factors (CSFs), including GM-CSF, G-CSF and IL-3, have been shown to be effective stimulators of both self-renewal and terminal differentiation of blast stem cells in acute myeloblastic leukemia (AML). We have examined the activity of a fourth growth factor, recombinant CSF-1 (or M-CSF), on the growth of leukemic blasts in culture. CSF-1 was found to be active on some, but not all, blast populations. In sensitive cells, CSF-1 often stimulated the production of adherent blast cells incapable of division. This observation leads us to suggest that CSF-1 may be useful in the treatment of selected cases of AML.