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Recently, the specific association between Sinonasal inverted papilloma (SIP) and EGFR exon 20 mutations has been reported. To investigate the link between specific EGFR mutations and SIP development, we established organotypic raft culture system using nasal polyp-derived immortalized NP2 (iNP2) cells expressing EGFR exon 20 mutants or an exon 19 mutant, and SIP-derived iIP4 cells harboring P772_H773insPYNP mutation. In the raft culture, iIP4 cells showed the inverted growth pattern characteristic to SIP. Interestingly, iNP2 cells expressing EGFR exon 20 duplication mutants, S768_D770dup and N771_H773dup, but not of EGFR exon 19 mutant, E746_A750del, showed the inverted growth pattern. Enhanced activation of the PI3K/AKT signaling pathway was observed in iNP2_S768_D770dup and iIP4 cells, while increased MAPK signaling was found in iNP2_N771_H773dup. Increased cell migration and invasion were found in all cells carrying EGFR mutations when compared to iNP2 cells, and this effect was inhibited by either PI3K or MEK inhibitor. Notably, iNP2 cells expressing the N771_H773dup mutant showed the highest migration and invasion abilities. These results suggest that specific mutations in EGFR exon 20 play a crucial role in SIP development, partially though hyper-activation of the PI3K/AKT and MAPK signaling pathways. This study presents the first in vitro model for SIP development, which could facilitate further investigations into SIP pathogenesis and preclinical studies for new therapeutic agents., (© 2023. The Author(s).)

To better understand the pathogenesis of nasal polyps (NPs) and sinonasal inverted papillomas (SIPs), we aimed to establish cell lines from fresh tissues of NPs and SIPs and characterize them. Primary cell cultures were obtained from two NP tissues (NP2 and NP3) and one SIP tissue (IP4). All the cells were polygonal in shape, expressed cytokeratin 14, and had normal diploid chromosome status. HPV58 DNA was detected in NP3. To obtain immortal primary cells, NP2 and IP4 cells were transduced with a combination of mutant CDK4, cyclinD1 and TERT. These cells were thereafter named NP2/K4DT and IP4/K4DT, respectively. HPV58-positive NP3 cells were transduced with TERT alone, the resulting cells named NP3/T. Phenotypic and genotypic identity of original tissues and derived cells was investigated. All the cell cultures with transgenes were confirmed to be derived from their parental cells and primary tumor tissues by analysis of short tandem repeats (STR) and maintained in vitro growth, genetic profiles and gene expression characteristics of the primary cells. These virtually immortalized cells, as well as the primary cells, have potential as in vitro models for studying the pathogenesis of NPs and SIPs and for preclinical study to develop new therapeutic agents., (© 2021. The Author(s).)

Ammonia has a cytotoxic effect and can therefore be used as a selection agent for enrichment of zone I hepatocytes. However, it has not yet been determined whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed an ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human embryonic stem cells (ESCs) and from induced pluripotent stem cells (iPSCs). The resistance to cytotoxicity or cell death by ammonia is likely attributable to the metabolism of ammonia in the cells. In addition to ammonia metabolism-related genes, ammonia-selected hepatocytes showed increased expression of the cytochrome P450 genes. Additionally, the ammonia-selected cells achieved immortality or at least an equivalent life span to human pluripotent stem cells without affecting expression of the liver-associated genes. Ammonia treatment in combination with in vitro propagation is useful for obtaining large quantities of hepatocytes.

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4 R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

Mitochondrial DNA (mtDNA) mutations are found in many types of cancers and suspected to be involved in carcinogenesis, although the mechanism has not been elucidated. In this study, we report that consecutive C-to-T mutations (hypermutations), a unique feature of mutations induced by APOBECs, are found in mtDNA from cervical dysplasia and oropharyngeal cancers. In vitro, we found that APOBEC3A (A3A) and 3B (A3B) expression, as well as mtDNA hypermutation, were induced in a cervical dysplastic cell line W12 when cultured in a differentiating condition. The ectopic expression of A3A or A3B was sufficient to hypermutate mtDNA. Fractionation of W12 cell lysates and immunocytochemical analysis revealed that A3A and A3B could be contained in mitochondrion. These results suggest that mtDNA hypermutation is induced upon keratinocyte differentiation, and shed light on its molecular mechanism, which involves A3s. The possible involvement of mtDNA hypermutations in carcinogenesis is also discussed.

Conservation of the genetic resources of endangered animals is crucial for future generations. The loggerhead sea turtle (Caretta caretta) is a critically endangered species, because of human hunting, hybridisation with other sea turtle species, and infectious diseases. In the present study, we established primary fibroblast cell lines from the loggerhead sea turtle, and showed its species specific chromosome number is 2n = 56, which is identical to that of the hawksbill and olive ridley sea turtles. We first showed that intensive hybridization among multiple sea turtle species caused due to the identical chromosome number, which allows existence of stable hybridization among the multiple sea turtle species. Expressions of human-derived mutant Cyclin-dependent kinase 4 (CDK4) and Cyclin D dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. The recombinant fibroblast cell lines maintained the normal chromosome condition and morphology, indicating that, at the G1/S phase, the machinery to control the cellular proliferation is evolutionally conserved among various vertebrates. To our knowledge, this study is the first to demonstrate the functional conservation to overcome the negative feedback system to limit the turn over of the cell cycle between mammalian and reptiles. Our cell culture method will enable the sharing of cells from critically endangered animals as research materials.

Copper (Cu) is an indispensable metal for normal development and function of humans, especially in central nervous system (CNS). However, its redox activity requires accurate Cu transport system. ATP7A, a main Cu(2+) transporting-ATPase, is necessary to efflux Cu across the plasma membrane and synthesize cuproenzymes. Menkes disease (MD) is caused by mutations in ATP7A gene. Clinically, MD is Cu deficiency syndrome and is treated with Cu-histidine injections soon after definite diagnosis. But outcome of the most remains poor. To estimate the standard therapy, Cu distribution in the treated classic MD patients is analyzed by synchrotron-generated X-ray fluorescence technique (SR-XRF), which identifies and quantifies an individual atom up to at subcellular level of resolution with wide detection area. SR-XRF analysis newly reveals that Cu exists in spinal cord parenchyma and flows out via venous and lymph systems. By systemic analysis, excess Cu is detected in the proximal tubular cells of the kidney, the mucosal epithelial cells of the intestine, and the lymph and venous systems. The current study suggests that the standard therapy supply almost enough Cu for patient tissues. But given Cu passes through the tissues to venous and lymph systems, or accumulate in the cells responsible for Cu absorption.

Cancer progression involves carcinogenesis, an increase in tumour size, and metastasis. Here, we investigated the effect of overexpressed CXC chemokine ligand 14 (CXCL14) on these processes by using CXCL14/BRAK (CXCL14) transgenic (Tg) mice. The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that for isogenic wild type C57BL/6 (Wt) mice. When tumour cells were injected into these mice, the size of the tumours that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumour cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity played an important role during CXCL14-mediated suppression of tumour growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed in B16 melanoma cells. Further, the survival rates after tumour cell injection were significantly increased for the Tg mice. As these Tg mice showed no obvious abnormality, we propose that CXCL14 to be a promising molecular target for cancer suppression/prevention.