Cooverexpression of alanine aminotransferase 1 in Chinese hamster ovary cells overexpressing taurine transporter further stimulates metabolism and enhances product yield.

Innovation in monoclonal antibody (mAb) production continues to be driven by cell engineering strategies to increase yield and improve product quality. In a previous study, to investigate the effectiveness of transporter overexpression strategies, we prepared a taurine transporter-overexpressing host cell line (DXB11/TAUT) that produced a higher proportion of high-mAb-titer strains than did the parent host cell line. In the current study, we selected a single DXB11/TAUT/mAb1 strain that remained viable for longer (up to 1 month) under common fed-batch culture conditions, and the improvement in viability could be attributed to its improved metabolic properties. It was also more productive (up to >100 pg/cell/day) and yielded more mAb1 (up to 8.1 g/L/31 days) than the parent cell line, and the mAb1 it produced was of comparable quality. These results suggested that this host cell engineering strategy has unique potential for the improvement of mAb-producing Chinese hamster ovary (CHO) cells; for example, it may be appropriate for high cell density perfusion culture. TAUT-overexpressing cell lines rapidly accumulated the byproduct alanine, and our challenge in the present study was to apply a strategy for modulating cell metabolism to utilize this byproduct to achieve a high mAb yield in a shorter culture period. To accomplish this, we genetically modified the DXB11/TAUT/mAb1 strain to cooverexpress alanine aminotransferase 1 (ALT1). The resulting DXB11/TAUT/mAb1/ALT1 cooverexpressing strain gave a higher mAb yield in a shorter culture period (5.9 g/L/14 days). It is usually difficult to drive the overexpression of two functional genes while balancing competing goals. However, forced cooverexpression of TAUT and ALT1 in our DXB11/TAUT/mAb1/ALT1 strain resulted in a higher proliferation than the DXB11/TAUT/mAb1 strain, with an ideal balance between cell viability and productivity. Therefore, we have demonstrated a strategy capable of achieving an optimum balance among the goals of cell viability, productivity, and proliferative capacity.

Overexpression of taurine transporter in Chinese hamster ovary cells can enhance cell viability and product yield, while promoting glutamine consumption.

Transporters mediate the uptake of nutrients such as amino acids and the excretion of metabolites. The fact that transporters play crucial roles in regulating cell metabolism suggests that they might be useful targets for cell engineering to enhance the yield and/or quality of monoclonal antibody (MAb) produced by CHO cells. The taurine transporter (TAUT) is stably expressed in CHO-DXB11 cells and is upregulated late in the culture period. We found that forcing the overexpression of TAUT delayed apoptotic cell death, extending the culture period. Thus, under fed-batch small-culture conditions, CHO cells that expressed pHyg-TAUT plasmid (TAUT/CHO cells), but not those that contained the null plasmid pHyg (HYG/CHO cells), produced more MAb (P < 0.01) and less lactate (P < 0.05). In a 1-L bioreactor, a representative high-yield TAUT/CHO cell line (T10) showed >80% viability for more than 1 month and a 47% increase in medium MAb concentration. In T10 cells, the upregulation of TNF-α mRNA (an apoptosis marker) and the accumulation of ammonia late in the culture period were suppressed. Moreover, if an excess of taurine was added, T10 cells efficiently consumed glutamine but not other amino acids, so T10 cells may have gained a glutamine transporter-like function. Because a considerable amount of metabolic energy is derived from glutamine, this active glutamine consumption in T10 cells might be a reason for the improved cell viability and MAb concentration. These results demonstrate that forcing the overexpression of TAUT in CHO cells can enhance cell culture performance and increase MAb titer.

5-Fluorouracil up-regulates interferon pathway gene expression in esophageal cancer cells.

5-Fluorouracil (FU) is a chemotherapeutic agent commonly used against esophageal cancer. Recently, interferons (IFNs) have been administered together with cytotoxic chemotherapy to patients with this cancer, although the mechanisms of synergy are unknown. We reconsidered the mechanisms for the effects of 5-FU in this context, aiming to refine combination therapy. After three cell lines (T.T, TE-2, and TE-6), derived from human esophageal squamous cell carcinoma (SCC), were exposed to 5-FU, the expression profiles were analyzed using high-density oligonucleotide microarrays representing about 12,000 genes. Among them, three IFN-related genes, an IFN receptor gene (IFNAR2) and two IFN-stimulated genes (ISG15K, ISG-54K), that were up-regulated following addition of 5-FU, were investigated. Up-regulation was confirmed by RT-PCR. Based on these results, the antitumor effects of exposure to 5-FU simultaneously with IFN-alpha, -beta and -gamma were investigated. The growth of esophageal SCC cells with 5-FU was suppressed synergistically or semi-additively by IFN-alpha and -beta, but not by IFN-gamma. These findings indicated that 5-FU stimulated IFN pathways in esophageal SCC cells following up-regulation of the IFN type I receptor. A combination of 5-FU and an IFN, therefore, may be particularly efficacious in esophageal cancer.

Gene expression analysis of renal carcinoma: adipose differentiation-related protein as a potential diagnostic and prognostic biomarker for clear-cell renal carcinoma.

The gene expression profiles of 33 renal cell carcinomas (RCCs) and nine normal kidney samples were examined using high-density oligonucleotide microarrays in an attempt to identify biomolecular markers for the diagnosis of tumour subtypes and also for prediction of prognosis. Hierarchical clustering demonstrated that clear-cell RCC, chromophobe RCC, and normal kidney tissue showed distinctive gene expression profiles. The mean expression levels of 149 of 12 500 genes were more than three times higher in clear-cell RCC than in chromophobe RCC and normal kidney tissue. Among the genes whose expression was upregulated in clear-cell RCC, adipose differentiation-related protein (ADFP) and nicotinamide N-methyltransferase (NNMT) were selected for further analysis. Consistent with the results of the microarray, increased levels of ADFP and NNMT mRNA were found more frequently in clear-cell RCCs than in other non-clear-cell tumour subtypes using real-time quantitative PCR. Immunohistochemistry for ADFP showed strong and unique tumour cell staining patterns in the majority of clear-cell RCCs. More importantly, patients bearing tumours with higher AFDP mRNA levels showed significantly better survival in both univariate and multivariate analyses. ADFP is a lipid storage droplet-associated protein and its transcription is considered to be regulated by the von Hippel-Lindau/hypoxia-inducible factor pathway. It is known that clear-cell RCC contains abundant lipids and cholesterols. Thus it is likely that sustained upregulation of ADFP following VHL inactivation is involved in the morphological appearance of clear-cell RCC. Moreover ADFP expression status may provide useful prognostic information as a biomolecular marker in patients with clear-cell RCC.

Loss of von Hippel-Lindau protein causes cell density dependent deregulation of CyclinD1 expression through hypoxia-inducible factor.

Loss of the von Hippel-Lindau gene (VHL) expression ca-uses deregulation of contact inhibition of cell growth, which might be one of the bases of the tumor suppressor function of VHL. Here we show that this function of the VHL gene product (pVHL) depends on cell autonomous events. To identify the target gene of pVHL, which is directly involved in the contact inhibition, we compared the gene expression profile between VHL-deficient renal carcinoma 786-O cells and those infected with an adenovirus vector encoding VHL. In addition to known pVHL-regulated genes, such as vascular endothelial growth factor and carbonic anhydrase, we found cyclinD1 as a new target of pVHL at a high cell density. In VHL-expressing cells (VHL (+) cells), the cyclinD1 mRNA expression level diminishes at a high cell density, while it remains at a relatively high level in VHL-deficient cells (VHL (-) cells). The cyclinD1 expression level was also abnormally high in VHL (-) cells at a high cell density. Consequently, the phosporylation level of the retinoblastoma (Rb) protein remained high in these cells, whereas there was no phosporylated Rb in VHL (+) cells under the contact inhibition. The abnormal expression of cyclinD1 at a high cell density was observed even in VHL (+) cells under the hypoxic state. Moreover, ectopic expression of a HIF mutant resistant to pVHL-mediated proteolysis causes the abnormal cyclinD1 expression in VHL (+) cells. Taken together, these observations indicate that VHL is required for the downregulation of cyclinD1 at a high cell density through HIF.

Oligonucleotide microarray for prediction of early intrahepatic recurrence of hepatocellular carcinoma after curative resection.

BACKGROUND: Hepatocellular carcinoma has a poor prognosis because of the high intrahepatic recurrence rate. There are technological limitations to traditional methods such as TNM staging for accurate prediction of recurrence, suggesting that new techniques are needed. METHODS: We investigated mRNA expression profiles in tissue specimens from a training set, comprising 33 patients with hepatocellular carcinoma, with high-density oligonucleotide microarrays representing about 6000 genes. We used this training set in a supervised learning manner to construct a predictive system, consisting of 12 genes, with the Fisher linear classifier. We then compared the predictive performance of our system with that of a predictive system with a support vector machine (SVM-based system) on a blinded set of samples from 27 newly enrolled patients. FINDINGS: Early intrahepatic recurrence within 1 year after curative surgery occurred in 12 (36%) and eight (30%) patients in the training and blinded sets, respectively. Our system correctly predicted early intrahepatic recurrence or non-recurrence in 25 (93%) of 27 samples in the blinded set and had a positive predictive value of 88% and a negative predictive value of 95%. By contrast, the SVM-based system predicted early intrahepatic recurrence or non-recurrence correctly in only 16 (60%) individuals in the blinded set, and the result yielded a positive predictive value of only 38% and a negative predictive value of 79%. INTERPRETATION: Our system predicted early intrahepatic recurrence or non-recurrence for patients with hepatocellular carcinoma much more accurately than the SVM-based system, suggesting that our system could serve as a new method for characterising the metastatic potential of hepatocellular carcinoma.