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Recombinant growth factors have been shown to alter the sensitivity of acute myeloblastic leukemia (AML) blast cells to cytosine arabinoside (ara-C) in culture. The mechanism is controversial and suggestions for it include changes in ara-C metabolism, changes in cell cycle parameters, and changes in the balance between self-renewal and determination in blast stem cells. We addressed this issue by measuring the cisplatin sensitivity of freshly obtained AML blasts in rG-CSF, rGM-CSF, or the two together. For comparison, simultaneous measurements of ara-C sensitivity were made. We found that exposure to different factors in suspension altered the cisplatin sensitivity of AML blasts in the same direction as the change observed in ara-C sensitivity. Similar changes in cisplatin sensitivity were seen when cells were briefly exposed to the drug, washed, and then grown in suspension in the presence of different growth factors. Control experiments showed that the conditions in suspension, not in the clonogenic assay in methylcellulose, were responsible for the changes in cisplatin sensitivity. The capacity of high specific activity to inactivate clonogenicity was tested at several times under growth conditions which altered the sensitivity of cells to cisplatin. Whereas changes in survival after 3HTdR and cisplatin both were seen with time, growth conditions that altered cisplatin sensitivity were not associated with changes in 3HTdR toxicity. The data do not support explanations of the effects of growth conditions on drug toxicity which depend either on drug metabolism or cell cycle effects. Instead, the findings are consistent with a model that postulates an association between drug sensitivity and the balance between self-renewal and differentiation in the blast population.

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.

Morphological-identified blast populations are the hallmark of the malignant clones that dominate hemopoiesis in acute myeloblastic leukemia (AML). Marked heterogenity is characteristic of AML blasts. Patient-to-patient variation is seen in their biological properties but is particularly evident in the response to treatment. Intraclonal variation is generated during clonal expansion, particularly as blast stem cells either undergo self-renewal or enter into a series of terminal divisions. These two alternative activities can be monitored in cell culture using a clonogenic assay and a suspension assay. The balance between renewal and differentiation can be altered by exposing blast populations to various growth factors in culture. Further, certain drugs, particularly ara-C, appear to be more toxic for self-renewing divisions than cell-cycle events generally. We suggest that both drugs and growth factors should be assessed for their effects on self-renewal as part of preclinical testing.

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.