Productivity enhancement of recombinant protein in CHO cells via specific promoter activation by oncogenes

To construct a recombinant protein highly producing cell lines, we have previously developed the Oncogene Activated Production (OAP) system by using BHK-21 cells. Here we verified the availability of the OAP system in CHO cells. We firstly generated ‘primed’ ras amplified CHO cells, ras clone I, by introducing human c-Ha-ras oncogene into CHO cells. This ras clone I enables quick and easy establishment of recombinant protein hyper producing cell lines by introduction reporter gene of interest. Then we generated I13 by introducing human interleukin 6 (hIL-6) gene as a reporter gene, which showed enhanced productivity rate as compared to A7 established by conventional method. Furthermore, we found that hIL-6 production level of I13 was slightly improved by raising the CO₂ concentration from 5 to 8% possibly because of the enhanced growth rate. We further introduced the E1A oncogene, which has been shown to have a synergistic effect on the recombinant protein production of the ras-amplified BHK-21 cells, then evaluated the productivity. When culture in 5% CO₂ condition, only the slight effect can be seen. However when cultured in 8% CO₂ condition, not only cell number, but also productivity increased significantly, resulted in great augmentation of hIL-6 production, maximum production being 88.6 μg/ml/3 days. This study demonstrates that recombinant protein production level reached commercially desirable level by utilizing our OAP system in CHO cells and optimizing the culture condition. This revised version was published online in August 2006 with corrections to the Cover Date.     

Structural and functional differences of the carotid body between DBA/2J and A/J strains of mice.

In a previous study, DBA/2J and A/J inbred mice showed extremely different hypoxic ventilatory responses, suggesting variations in their carotid bodies. We have assessed the morphological and functional differences of the carotid bodies in these mice. Histological examination revealed a clearly delineated carotid body only in the DBA/2J mice. Many typical glomus cells and glomeruli appeared in the DBA/2J but not in the A/J mice. The size of the carotid body in the DBA/2J and A/J mice was 6.3 +/- 0.5 x 10(6) and 1.5 +/- 0.3 x 10(6) micro m(3), respectively. The area immunostained for tyrosine hydroxylase, an estimation of the glomus cell quantity, was four times larger in the DBA/2J mice than in the A/J mice. The individual data points in the DBA/2J mice segregated from those in the A/J mice. ACh increased intracellular Ca(2+) in most clusters (81%) of cultured carotid body cells from the DBA/2J mice, but only in 18% of clusters in the A/J mice. These data suggest that genetic determinants account for the strain differences in the structure and function of the carotid body.     

Monospecific antiserum to rat spermidine synthase and its application to rat tissues and several mammals

Monospecific antiserum to rat spermidine synthase was prepared by immunization of rabbits with purified enzyme protein from rat prostate, and its usefulness for analysis of spermidine synthase protein in not only rat tissues but also several other mammals was demonstrated by Western blotting and immunotitration of the enzyme activity. Application of the antiserum for elucidating the relationship between the enzyme activity and protein in normal rat tissues strongly suggested that marked difference in spermidine synthase activity among rat tissues depends solely on the difference in the amount of enzyme protein. Also, application of the antiserum for analyzing spermidine synthase from liver of mouse, rat, guinea pig, pig, and human, showed that the enzymes had a similar subunit molecular weight of 35,000 and a cross-reactivity with the antiserum, exhibiting almost the same immunoreactivity to mouse enzyme as to rat enzyme. Thus, it was suggested that the antiserum would be useful for further studies of mammalian spermidine synthase from the viewpoints of enzymology and molecular biology.     

Suppressive effects of electrochemically reduced water on matrix metalloproteinase-2 activities and in vitro invasion of human fibrosarcoma HT1080 cells

It has been demonstrated that hydrogen peroxide (H(2)O(2)) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H(2)O(2) in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H(2)O(2)-induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH(2)-terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H(2)O(2)-treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect.     

A potential estrogen mimetic effect of a bis(ethyl)polyamine analogue on estrogen receptor positive MCF-7 breast cancer cells

BE-3-3-3-3 (1,15-(ethylamino)4,8,12-triazapentadecane) is a bis(ethyl)polyamine analogue under investigation as a therapeutic agent for breast cancer. Since estradiol (E(2)) is a critical regulatory molecule in the growth of breast cancer, we examined the effect of BE-3-3-3-3 on estrogen receptor α (ERα) positive MCF-7 cells in the presence and absence of E(2). In the presence of E(2), a concentration-dependent decrease in DNA synthesis was observed using [(3)H]-thymidine incorporation assay. In the absence of E(2), low concentrations (2.5-10 μM) of BE-3-3-3-3 increased [(3)H]-thymidine incorporation at 24 and 48 h. BE-3-3-3-3 induced the expression of early response genes, c-myc and c-fos, in the absence of E(2), but not in its presence, as determined by real-time quantitative polymerase chain reaction (qPCR). BE-3-3-3-3 had no significant effect on these genes in an ERα-negative cell line, MDA-MB-231. Chromatin immunoprecipitation assay demonstrated enhanced promoter occupation by either E(2) or BE-3-3-3-3 of an estrogen-responsive gene pS2/Tff1 by ERα and its co-activator, steroid receptor co-activator 3 (SRC-3). Confocal microscopy of BE-3-3-3-3-treated cells revealed membrane localization of ERα, similar to that induced by E(2). The failure of BE-3-3-3-3 to inhibit cell proliferation was associated with autophagic vacuole formation, and the induction of Beclin 1 and MAP LC3 II. These results indicate a differential effect of BE-3-3-3-3 on MCF-7 cells in the absence and presence of E(2), and suggest that pre-clinical and clinical development of polyamine analogues might require special precautions and selection of sensitive subpopulation of patients.     

Building high affinity human antibodies by altering the glycosylation on the light chain variable region in N-acetylglucosamine-supplemented hybridoma cultures

We attempted to improve antibody affinity by varying glycosylation on the light chain variable region. The human hybridoma line HB4C5 produces an antibody reactive to lung adenocarcinoma, which possess a N-glycosylated carbohydrate chain on the light chain hypervariable region. It has been shown that altering this carbohydrate structure can be accomplished by varying the level of N-acetylglucosamine in glucose free medium, a change in the carbohydrate chain could be induced which resulted in modifying antigen binding. By culturing the cells in media containing more than 20 mM N-acetylglucosamine, cells produced antibody with 10 fold improved affinity as compared with antibody produced in 20 mM glucose-containing medium. A newly induced light chain glycoform produced in the N-acetylglucosamine-containing medium was shown to be responsible for this antigen binding enhancement. Addition of glucose in the N-acetylglucosamine-containing media led to decreased antibody affinity and slightly inhibited production of a new light chain in a dose-dependent manner. Combination of 20 mM N-acetylglucosamine and 0.5 mM glucose gave a higher antibody production without the decrease of the antigen binding. These results indicate that optimization of N-glycosylation on the light chain, which leads to higher antigen binding, can be accomplished by adjusting a ratio of glucose and N-acetylglucosamine in the culture medium.     

Analysis of comorbid factors that increase the COPD assessment test scores

Background

The chronic obstructive pulmonary disease (COPD) Assessment Test (CAT) is a concise health status measure for COPD. COPD patients have a variety of comorbidities, but little is known about their impact on quality of life. This study was designed to investigate comorbid factors that may contribute to high CAT scores.

Methods

An observational study at Keio University and affiliated hospitals enrolled 336 COPD patients and 67 non-COPD subjects. Health status was assessed by the CAT, the St. Georges Respiratory Questionnaire (SGRQ), and all components of the Medical Outcomes Study Short-Form 36-Item (SF-36) version 2, which is a generic measure of health. Comorbidities were identified based on patients’ reports, physicians’ records, and questionnaires, including the Frequency Scale for the Symptoms of Gastro-esophageal reflux disease (GERD) and the Hospital Anxiety and Depression Scale. Dual X-ray absorptiometry measurements of bone mineral density were performed.

Results

The CAT showed moderate-good correlations with the SGRQ and all components of the SF-36. The presence of GERD, depression, arrhythmia, and anxiety was significantly associated with a high CAT score in the COPD patients.

Conclusions

Symptomatic COPD patients have a high prevalence of comorbidities. A high CAT score should alert the clinician to a higher likelihood of certain comorbidities such as GERD and depression, because these diseases may co-exist unrecognized.

Trial registration

Clinical trial registered with UMIN (UMIN000003470).