Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

Miyaji, Hiromasa; Harada, Nahoko; Mizukami, Tamio; Sato, Seiji; Fujiyoshi, Nobuo; Itoh, Seiga

doi:10.1007/bf00148809

Cited by 8 publications

(1 citation statement)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…MC-9, Ba/F-3, DA-1, IC-2, and FDCP mix, was washed 3 times with the medium, and seeded on the stromal cell layer at a concentration of 3.5 x 104 cells/ml in 100 pi of the medium. Two controls were set; one consisted of MMC-treated stromal cells alone, and the other con- into NFS-60 cells, using the electroporation method described pre viously [30]. 1.6 x 106 cells suspended in 44 pi of phosphate-buff ered saline containing 4 pg of pfosluc2 were subjected to electropora tion in high electric fields using the somatic hybridizer SSH-1 (Shimadzu Seisakusyo, Kyoto, Japan).…”

Section: Coculture Of Myeloid Cell Lines and Stromal Cell Line Pa6mentioning

confidence: 99%

Characterization of the Molecules Involved in the Hematopoietic Microenvironment Provided by Mouse Stromal Cell Line MC3T3-G2/PA6 Using a Unique Reporter System That Analyzes the Direct Cell-to-Cell Interaction

Satoh¹,

Mioh²,

Konishi³

et al. 1997

Acta Haematol

View full text Add to dashboard Cite

As an approach to characterizing the molecules involved in the hematopoietic microenvironment provided by a murine clonal preadipose cell line MC3T3-G2/PA6 (PA6), we developed a unique system to detect the early phase of signal transduction caused by the direct cell-to-cell interaction using the reporter plasmid pfosluc2 with the c-fos enhancer/promoter linked with the Photinus pyralis luciferase gene. The plasmid pfosluc2 was genetically introduced into a mouse myeloid leukemia cell line NFS-60 which showed a growth dependency on contact with PA6 cells, and the mechanism by which stromal PA6 cells promote the proliferation of NFS-60 cells through the direct cell-to-cell interaction was analyzed. The direct cell-to-cell interaction with PA6 cells was found to cause a significant c-fos induction to NFS-60 cells within 1 h. Approximately 105 cDNA clones prepared from PA6 cells were screened for their activity to promote the c-fos expression in NFS-60 cells through the direct cell-to-cell interaction, and 13 positive clones were obtained. Of these positive clones, five clones encoded the stem cell factor, and the others encoded the hepatocyte growth factor (HGF). The c-fos induction caused by the contact with PA6 cells in NFS-60 was completely inhibited by addition of both antagonistic anti-c-kit and anti-HGF antibodies. These results represent direct evidence for the action of HGF on the proliferation of hematopoietic cells through direct cell-to-cell interaction with stromal cells. Thus, our developed reporter system can be useful in investigating the direct cell-to-cell interaction between stromal and hematopoietic cells.

show abstract

Section: Coculture Of Myeloid Cell Lines and Stromal Cell Line Pa6mentioning

confidence: 99%

Characterization of the Molecules Involved in the Hematopoietic Microenvironment Provided by Mouse Stromal Cell Line MC3T3-G2/PA6 Using a Unique Reporter System That Analyzes the Direct Cell-to-Cell Interaction

Satoh¹,

Mioh²,

Konishi³

et al. 1997

Acta Haematol

View full text Add to dashboard Cite

show abstract

Optimization of cell culture conditions for G-CSF (granulocyte colony-stimulating factor) production by genetically engineered Namalwa KJM-1 cells

Hosoi

Murosumi

Sasaki

et al. 1991

Animal Cell Culture and Production of Biologicals

View full text Add to dashboard Cite

Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered namalwa KJM-1 cells

et al. 1991

Self Cite

View full text Add to dashboard Cite

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 micrograms/ml/day using a 25 cm2 tissue culture flask, even though the cell number was above 7 x 10(5) cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 4 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1 x 10(7) cells/ml), and the amount of G-CSF reached 41 micrograms/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.

show abstract

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

Cited by 8 publications

References 12 publications

Characterization of the Molecules Involved in the Hematopoietic Microenvironment Provided by Mouse Stromal Cell Line MC3T3-G2/PA6 Using a Unique Reporter System That Analyzes the Direct Cell-to-Cell Interaction

Characterization of the Molecules Involved in the Hematopoietic Microenvironment Provided by Mouse Stromal Cell Line MC3T3-G2/PA6 Using a Unique Reporter System That Analyzes the Direct Cell-to-Cell Interaction

Optimization of cell culture conditions for G-CSF (granulocyte colony-stimulating factor) production by genetically engineered Namalwa KJM-1 cells

Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered namalwa KJM-1 cells

Contact Info

Product

Resources

About