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Cell proliferation in the bone marrow and blood of two patients with metastatic breast cancer who were treated with granulocyte colony-stimulating factor was studied by using [3H]thymidine labeling and autoradiography. Additionally, the fate of neutrophils labeled with "Tc-hexamethylpropyleneamineoxime was observed following granulocyte colony-stimulating factor infusion. Proliferation increased in all stages of granulopoiesis, but a significant amount of the increased production stemmed from a greater input to the myeloblast compartment. Changes in the myelogram combined with the increased labeling indicated a faster throughput of cells, which resulted in labeled cells appearing in the circulation within 1 day compared to the normal 4 or 5 days. The ""'Tc studies demonstrated no sequestration of circulating neutrophils by spleen, lungs, or liver. The halflife of the circulating neutrophils was not significantly changed, and calculations from the flow of labeled cells to the peripheral blood indicated an increase of 3.2 extra amplification divisions during neutrophil development. The dramatic neutrophil response to granulocyte colony-stimulating factor can therefore be accommodated by a relatively modest increase in granulopoietic activity.The recent availability of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (1) has stimulated considerable interest in its potential applications for promoting hemopoietic regeneration following bone marrow damage by cytoreductive agents. Stimulation, in vivo, of granulopoiesis by rhG-CSF was demonstrated in mice (2) and in humans (3-7). In patients, 2 or 3 days of continuous infusion or repeated injections of rhG-CSF resulted in a 10-fold increase in peripheral neutrophil levels, which were maintained for the duration of infusion and, following chemotherapy, significantly shortened the duration of the subsequent neutropenia (3,(5)(6)(7). These neutrophils demonstrated normal function and competence (4). Only a small, nonsignificant increase in granulocyte/macrophage colony-forming cell (GM-CFC) cycling and in the GM-CFC/burst-forming unit-erythroid ratio was observed, suggesting that most of the expansion in neutrophil production probably arises after the GM-CFC stage (4). Our intention was to investigate the changes in kinetics induced by G-CSF and required to maintain this elevated neutrophilia. We therefore treated two patients with rhG-CSF and, by means of autoradiography, studied the kinetics of marrow cell proliferation and efflux into the peripheral blood following labeling in vivo with tritiumlabeled thymidine. Since rhG-CSF in vivo initially produces an early fall in peripheral neutrophils followed by rapid influx of mature neutrophils into the circulatory pool (4), we also studied the early effects of this growth factor on circulating neutrophils by labeling them with 99mTc-hexamethylpropyleneamineoxime (9mTc-HMPAO) (8) and observing their fate with a y camera. MATERIALS AND METHODSPatients and Therapy. Two patients with metastatic breast c...

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.

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