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We compared the antiapoptotic activity of a recombinant chimera of insulin-like growth factor II (IGF-II) and interleukin (IL)-3 with the corresponding equimolar mixture of the individual components based on changes in several factors associated with survival in the CD34+ human hematopoietic cell line TF-1. Propidium iodide-stained cells analyzed by fluorescein-activated cell sorter indicated that the chimera was more effective than the corresponding equimolar mixture in decreasing the amounts of apoptotic cells and increasing the proportion of cells in the S-phase of the cell cycle. The chimera was more effective in increasing the antiapoptotic protein Bclx(L) and produced a significant increase in signal transducer and activator of transcription-5 phosphorylation and in phosphatidylinositol-3 kinase (PI-3K) activity. The PI-3K inhibitor LY294002 specifically inhibited cell survival in the presence of the chimera, suggesting a key role of this enzyme in the potentiation of survival caused by the linkage of IGF and IL-3. This potentiation of survival and its preferential inhibition by LY294002 were also observed in a nontransformed, primary culture of human umbilical cord endothelial cells.

Alpha(1)-proteinase inhibitor (API) was coupled at the C-terminus of a human insulin-like growth factor (IGF) analog to facilitate its production in insect cells. This fusion protein significantly increased thymidine incorporation into HL-60 cells as compared with the incorporation observed with an equivalent molar mixture of the IGF analog and API. The M351E variant of API has been previously shown to reduce aggregate formation in prokaryotic expression systems. When the oxidation-sensitive methionine 351 of the inhibitor was changed to glutamate, the M351E variant was secreted in larger amounts from insect cells than the corresponding fusion protein with wild-type API. The M351E fusion protein and the corresponding chimera containing the wild-type API were tested for their capacity to inhibit human neutrophil elastase. The M351E variant was a more potent elastase inhibitor than the fusion protein containing the wild-type analog, whereas the proliferative activity of both chimeras was identical. The described mitogenic effect of the chimera and the improved anti-elastase activity of the M351E variant are two ideal properties for therapeutic agents acting in pathological situations where cell proliferation and inhibition of neutrophil elastase have to take place simultaneously, such as during wound healing.

The erythropoietic factors present in an anephric patient with nearly normal hematocrit were isolated from plasma by reversed-phase and gel permeation HPLC. The most active fraction was purified and the analysis of its N-terminal sequence was identical to the published sequence of the human insulin-like growth factor I (IGF I). Recombinant human IGF I had identical elution positions as the isolated erythropoietic factor on reversed-phase HPLC and the same molecular weight on gel permeation HPLC. Furthermore, hrIGF I stimulated erythroid colony formation in human bone marrow cultures as was previously shown for the isolated human erythropoietic factor. These results suggest that IGF I may replace erythropoietin as a stimulator of erythropoiesis in some patients with anemia and renal failure.

The role of isoproterenol in the synthesis of heme associated with hemoglobin has been studied in primary cultures of human fetal liver. This drug (10-10M) stimulated the incorporation of 59Fe into heme associated with hemoglobin in cell cultures of livers obtained from human fetuses 8 to 10 weeks gestation but was inactive in cell cultures prepared from livers of older fetuses. Isoproterenol has its optimal activity at a much earlier time in gestation than that previously reported for testosterone or erythropoietin. The interpretation of these results is that there is present a cell population in human fetal liver early in gestation with the appropriate isoproterenol-receptors and capable of erythroid differentiation after exposure to the drug.