The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 lg 10 À6 cells day À1 , one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-jB in D29 cells. These results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the effect of pyrrolidinedithiocarbamate, an inhibitor of NF-jB activation, was examined. Suppression of NF-jB activation in D29 cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the release of negative feedback signals could increase the total amount of recombinant protein secretion.Abbreviations: CHO -Chinese hamster ovary; dhfr -dihydrofolate reductase; DMEM -Dulbecco's modified Eagle's medium; ECL -enhanced chemiluminescence; ELISA -enzyme-linked immunosorbent assay; EMSA -electrophoretic mobility shift assay;

We previously developed a promoter-activated production (PAP) system using amplified ras oncogene to activate the cytomegalovirus (CMV) promoter controlling the foreign gene in mammalian cells. CHO cells were demonstrated to be suitable for the PAP system. Here, we show that very high-level production of a recombinant protein was achieved when the human CMV promoter was inserted into a glutamine synthetase (GS) minigene expression plasmid, pEE14. A highly productive host CHO cell line, ras clone I containing amplified ras oncogene, was further transfected with the plasmid expressing both hIL-6 gene and GS minigene, and selected with methionine sulphoximine. We were able to establish a hIL-6 hyperproducing cell line, D29, which exhibited a peak productivity rate of approximately 40 lg hIL-6 10 À6 cells day À1 through a combination of the PAP system and the GS gene amplification system. The cellular productivity of D29 cells was about 13-fold higher than control hIL-6-producing cells derived from CHO cells whose hIL-6 gene was amplified by the GS gene amplification system, and about 5-fold higher than the I13 cells established by the PAP system, which contains amplified ras oncogene and nonamplified hIL-6 gene. When D29 cells were cultured for a month, an accumulation rate of approximately 80 lg hIL-6 ml À1 per 3 days was achieved on the 9th day. These results indicate that this PAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.Abbreviations: CHO -Chinese hamster ovary; CMV -cytomegalovirus; dhfr -dihydrofolate reductase; DMEM -Dulbecco's modified Eagle's medium; ELISA -enzyme-linked immunosorbent assay; FBS -fetal bovine serum; GS -glutamine synthetase; MSX -methionine sulphoximine; MTX -methotrexate; PAP system -promoter-activated production system.

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著者抄録：国内の科学技術誌や国内学会の要旨集などの定期刊行性のあ る 「国内科学誌」 を収録対象 と する学術情報データベース と し ては， JSTPlus 1) (科学技術振興機構)， JMEDPlus 2) (科学技術振興 機構)， 医学中央雑誌 3) (医学中央雑誌刊行会)， CiNii 4) (国立情報学研究所)， JAPICDOC 5) (日本医 薬情報セン ター) ， 医学薬学予稿集 (科学技術振興機構) ， J-STAGE (科学技術振興機構) ， Medical Online (Meteointergate Inc.

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