Masahiko Tamura

Molecular Cloning and Expression of Megakaryocyte Potentiating Factor cDNA

The human megakaryocyte potentiating factor (hMPF) has been previously purified from a culture supernatant of human pancreatic cancer cells HPC-Y5 (Yamaguchi, N., Hattori, K., Oh-eda, M., Kojima, T., Imai, N., and Ochi, N.(1994) J. Biol. Chem. 269, 805-808). We have now isolated hMPF cDNA from a HPC-Y5 cDNA library using polymerase chain reaction and plaque hybridization methods. The hMPF cDNA encodes a polypeptide consisting of 622 amino acids, including a signal peptide of 33 amino acids, and with a deduced molecular mass of 68 kDa, although HPC-Y5 cells secrete a 33-kDa form of hMPF. Human MPF does not show any significant homology with other previously described sequences. The cDNA was expressed in COS-7 and Chinese hamster ovary (CHO) cells, and megakaryocyte potentiating activity was detected in their culture supernatant. The COS-7 cells secreted only a 33-kDa recombinant hMPF, whereas an additional 30-kDa form was detected in the culture medium of CHO cells. The 33-kDa rhMPF purified from CHO cells showed megakaryocyte potentiating activity, but not the purified 30-kDa rhMPF. The difference in structure and activity between the 33- and 30-kDa forms of hMPF was ascribed to the existence in the 33-kDa form of the C-terminal 25 amino acid residues.

Induction of neutrophilic granulocytosis in mice by administration of purified human native granulocyte colony-stimulating factor (G-CSF)

Mice were subcutaneously (sc) injected once a day for up to 15 days with a purified human native G-CSF sample at a dose of 2.5 micrograms/injection or with control samples with or without added endotoxin. In the G-CSF-treated mice, blood neutrophil counts began to rise as early as 2 hours after the first injection, reached a level 8 times above the preinjection level after 15 days of injections with marked elevation of all progenitor cell levels in spleen, and returned to normal within 48 hours after cessation of the injections. Such neutrophilia was observed even when endotoxin-resistant C3H/HeJ mice were used, but not in control mice. It is possible that repeated G-CSF injections after administration of cyclophosphamide (CY) in mice could accelerate recovery of granulopoiesis with a rather transient rise in blood neutrophil counts.

Mechanism of Protective Effect of Recombinant Human Granulocyte Colony-Stimulating Factor (rG-CSF) on Pseudomonas Infection

Decrease in resistance to systemic Pseudomonas infection in cyclophosphamide (CPA)‐induced neutropenic mice was prevented by injections of recombinant human granulocyte colony‐stimulating factor (rG‐CSF). In order to explore mechanism of the prevention of CPA‐induced decrease in the anti‐infectious resistance by rG‐CSF, CPA‐treated and then rG‐CSF‐injected mice were inoculated i.p. with P. aeruginosa, and growth of the infecting bacteria and infiltration of leukocytes in the peritoneal cavity were determined. In the mice who had received 4 daily s.c. injections of rG‐CSF from the day after CPA‐injection, a large number of neutrophils were mobilized into the peritoneal cavity in response to the bacterial inoculation and growth of the infecting Pseudomonas in the cavity was markedly‐inhibited, whereas in CPA‐induced neutropenic mice few neutrophils were mobilized and the infecting bacteria proliferated vigorously in the peritoneal cavity. These results suggest that administration of rG‐CSF prevents CPA‐induced neutropenia and neutrophils circulating at normal level in the number are normally mobilized into the peritoneal cavity in response to Pseudomonas inoculation, and that the mobilized neutrophils inhibit proliferation of the infecting Pseudomonas.

Effects of recombinant human granulocyte colony-stimulating factor on neutrophil functions in aged animals

We have studied the effects of recombinant human granulocyte colony‐stimulating factor (rG‐CSF) on granulopoiesis and neutrophil functions in aged rats and aged mice. We subcutaneously injected rG‐CSF or control vehicle into aged rats (22 months old and 25 months old) for 7 consecutive days. counted the peripheral neutrophils and evaluated the functions of neutrophils isolated from venous blood. The peripheral neutrophil count in aged rats tended to be increased as compared with that in young rats (11 weeks old). However, the neutrophils in aged rats exhibited a decline of superoxide anion (O2‐‐) release and phagocytic activity as compared with young rats. The peripheral neutrophil count in aged rats was significantly increased 5–6‐fold as many as the control value by rG‐CSF treatment, which was accompanied by a significant enhancement of O2‐release and of phagocytic activity being restored to normal levels or better.

Prolonged survival of mice with myeloid leukemia by subcutaneous injection of recombinant human G-CSF

We studied the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on leukemia development and survival of mice with leukemia by using a radiation-induced myeloid leukemia cell line (C2M) with A-type phosphoglycerate kinase (PGK) as marker isoenzyme. C3H/He mice with B-type PGK were inoculated with 2 x 10(6) C2M cells through the tail vein. From the next day, they received a daily subcutaneous injection of 1 microgram rhG-CSF or control solution. The survival of rhG-CSF-treated recipients of C2M was significantly longer than control-solution-treated recipients. In rhG-CSF-treated recipients, not only spleen weight but also the number of blasts in hemopoietic organs was less than those in control recipients. While there was a remarkable decrease in bone marrow content of CFU-GM in control recipients, the content in rhG-CSF-treated recipients was comparable to that in normal mice. A reduced bone marrow and spleen content of leukemic colony-forming cells (L-CFU) was observed in rhG-CSF-treated recipients in comparison with control recipients. The electrophoretic analysis of PGK phenotypes of hemopoietic organs indicated the delayed appearance of A-type PGK from C2M cells in rhG-CSF-treated recipients. From these findings, we concluded that the injection of rhG-CSF delayed the onset of leukemia and improved the survival and several hematological parameters by suppressing C2M cells in recipient mice.

Reduction of the leukemogenic potential of malignant murine leukemic cells by in vivo treatment with recombinant human granulocyte colony-stimulating factor

We have investigated the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration on the leukemogenic potential of L-103 murine leukemic cells. Leukemogenic potential was assessed by comparing the regression lines drawn between the number of inoculated leukemic spleen cells and the mean survival time (MST) of the syngeneic recipients. rhG-CSF injected 2.5 micrograms daily for 14 days reduced the leukemogenic potential of spleen cells of the leukemic mice to 1/200 of the control. This phenomenon was not observed with the leukemic spleen cells treated with r-murine granulocyte-macrophage (rmGM)-CSF in vivo. Cytochemical study indicated that morphologically identifiable blast cells were fewer in the rhG-CSF-treated leukemic spleen. Furthermore, leukemic cells in the rhG-CSF-treated spleen were less proliferative than the control in spite of having more clonogenic cells in the leukemic cell preparation. Cytogenetical analysis showed that chromosome abnormalities found in the original leukemic cells were not altered by rhG-CSF administrations. It also showed that the frequency of the abnormal karyotype was reduced in rhG-CSF-treated leukemic spleen (4/17) as compared with the control (8/8), indicating that the mitotic fraction was smaller in the rhG-CSF-treated leukemic cells. These findings indicate that in addition to the reduced number of leukemic cells in the spleen cell preparation, a reduction of the proliferative capacity of the original leukemic cells is involved in the reduction of leukemogenic potential of leukemic cells treated with rhG-CSF in vivo.

Acceleration of the hemopoietic reconstitution in mice undergoing bone marrow transplantation by recombinant human granulocyte colony-stimulating factor

We have investigated the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on hemopoietic reconstitution after bone marrow transplantation (BMT) following lethal irradiation in mice. Mice received a daily administration of 10 μg/kg rG-CSF or control vehicle one through 21 days after BMT. Spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte colony-forming units (CFU-Meg), and erythroid burst-forming units (BFU-E) increased in both bone marrow and spleen of the rG-CSF-treated mice as compared with the control. This increase was evident during the administration period. In spite of the increase in the progenitor cells in bone marrow and spleen, only a recovery of neutrophils was accelerated in peripheral blood. Thus rG-CSF accelerated granulopoietic recovery in the BMT mice, with an enhanced recovery of the stem cells and the progenitors for erythrocytes and megakaryocytes. These results indicate the potential clinical usefulness of rG-CSF in the treatment of patients undergoing BMT.