Effects of genome instability of parental CHO cell clones on chromosome number distribution and recombinant protein production in parent-derived subclones.

Chinese hamster ovary (CHO) cells are the de facto standard host cells for biopharmaceuticals, and there is great interest in developing methods for constructing stable production cell lines. In this study, clones with a wide chromosome number distribution were selected from isolated antibody-producing strains, and subclones obtained from these clones were evaluated. The transgene copy number varied between the subclones. Even among subclones with similar copy numbers of antibody genes and maintained insertion sites, clones with different productivity were generated. Although the chromosome number distribution differed between these subclones, there was no correlation between the variability in chromosome number after cloning (genome instability) and productivity. Most of the subclones obtained from a parental strain with a wide chromosome number had the same wide chromosome number distribution as the parental strain. Less frequently, cells with less variation (remaining in one distribution) in chromosome number were isolated from cells with a wide chromosome number distribution, from which subclones with less variation in chromosome number were obtained when subcloning was performed again. These results imply that the characteristics of clones with chromosomal instability are inherited by subclones, and thus provide a better understanding of cell line stability/instability.

Beclin1 is essential for the pancreas development.

Beclin1 (Becn1) is a multifunctional protein involved in autophagy regulation, membrane trafficking, and tumor suppression. In this study, we examined the roles of Becn1 in the pancreas development by generating mice with conditional deletion of Becn1 in the pancreas using pancreatic transcriptional factor 1a (Ptf1a)-Cre mice (Becn1f/f; Ptf1aCre/+). Surprisingly, loss of Becn1 in the pancreas resulted in severe pancreatic developmental defects, leading to insufficient exocrine and endocrine pancreatic function. Approximately half of Becn1f/f; Ptf1aCre/+ mice died immediately after birth. However, duodenum and neural tissue development were almost normal, indicating that pancreatic insufficiency was the cause of death. These findings demonstrated a novel role for Becn1 in pancreas morphogenesis, differentiation, and growth, and suggested that loss of this factor leaded to pancreatic agenesis at birth.

Measurements of the lateral cerebellar hemispheres using near-infrared spectroscopy through comparison between autism spectrum disorder and typical development.

The cerebellum plays a vital role in cognition, communication with the cerebral cortex, and fine motor coordination. Near-infrared spectroscopy (NIRS) is a portable, less restrictive, and noninvasive functional brain imaging method that can capture brain activity during movements by measuring the relative oxyhemoglobin (oxy-Hb) concentrations in the blood. However, the feasibility of using NIRS to measure cerebellar activity requires discussion. We compared NIRS responses between areas assumed to be the cerebellum and the occipital lobe during a fine motor task (tying a bow knot) and a visual task. Our results showed that the oxy-Hb concentration increased more in the occipital lobe than in the cerebellum during the visual task (p =.034). In contrast, during the fine motor task, the oxy-Hb concentration decreased in the occipital lobe but increased significantly in the cerebellum, indicating a notable difference (p =.015). These findings suggest that we successfully captured cerebellar activity associated with processing, particularly fine motor coordination. Moreover, the observed responses did not differ between individuals with autism spectrum disorder and those with typical development. Our study demonstrates the meaningful utility of NIRS as a method for measuring cerebellar activity during movements.

Artificial induction of chromosome aneuploidy in CHO cells alters their function as host cells.

Chinese hamster ovary (CHO) cells are major host cells for biopharmaceuticals. During culture, the chromosome number of CHO cells alters spontaneously. Here, we investigated the effects of artificial changes in the chromosome number on productivity. When cell fusion between antibody-producing CHO-K1-derived cells was induced, we observed a wide range of aneuploidy that was not detected in controls. In particular, antibody productivities were high in clone-derived cell populations that retained a diverse chromosome number distribution. We also induced aneuploid cells using 3-aminobenzamide that causes chromosome non-disjunction. After induction of aneuploidy by 3-aminobenzamide, cells with an increased chromosome number were isolated, but cells with a decreased chromosome number could not be isolated. When antibody expression vectors were introduced into these isolated clones, productivity tended to increase in cells with an increased chromosome number. Further analysis was carried out by focusing on clone 5E8 with an average chromosome number of 37. When 5E8 cells were used as host, the productivity of multiple antibodies, including difficult-to-express antibodies, was improved compared with CHO-K1 cells. The copies of exogenous genes integrated into the genome were significantly increased in 5E8 cells. These findings expand the possibilities for host cell selection and contribute to the efficient construction of cell lines for recombinant protein production.

Radiosensitizing Effect of TRPV1 Channel Inhibitors in Cancer Cells.

Radiosensitizers are used in cancer therapy to increase the γ-irradiation susceptibility of cancer cells, including radioresistant hypoxic cancer cells within solid tumors, so that radiotherapy can be applied at doses sufficiently low to minimize damage to adjacent normal tissues. Radiation-induced DNA damage is repaired by multiple repair systems, and therefore these systems are potential targets for radiosensitizers. We recently reported that the transient receptor potential vanilloid type 1 (TRPV1) channel is involved in early responses to DNA damage after γ-irradiation of human lung adenocarcinoma A549 cells. Therefore, we hypothesized that TRPV1 channel inhibitors would have a radiosensitizing effect by blocking repair of radiation-induced cell damage. Here, we show that pretreatment of A549 cells with the TRPV1 channel inhibitors capsazepine, AMG9810, SB366791 and BCTC suppressed the γ-ray-induced activation of early DNA damage responses, i.e., activation of the protein kinase ataxia-telangiectasia mutated (ATM) and accumulation of p53-binding protein 1 (53BP1). Further, the decrease of survival fraction at one week after γ-irradiation (2.0 Gy) was enhanced by pretreatment of cells with these inhibitors. On the other hand, inhibitor pretreatment did not affect cell viability, the number of apoptotic or necrotic cells, or DNA synthesis at 24 h after irradiation. These results suggest that inhibition of DNA repair by TRPV1 channel inhibitors in irradiated A549 cells caused gradual loss of proliferative ability, rather than acute facilitation of apoptosis or necrosis. TRPV1 channel inhibitors could be novel candidates for radiosensitizers to improve the efficacy of radiation therapy, either alone or in combination with other types of radiosensitizers.