Association between KRAS and PIK3CA Mutations and Progesterone Resistance in Endometriotic Epithelial Cell Line.

Although endometriosis is a benign disease, it is associated with cancer-related gene mutations, such as KRAS or PIK3CA. Endometriosis is associated with elevated levels of inflammatory factors that cause severe pain. In a previous study, we demonstrated that KRAS or PIK3CA mutations are associated with the activation of cell proliferation, migration, and invasion in a patient-derived immortalized endometriotic cell line, HMOsisEC10. In this study, we investigated the effects of these mutations on progesterone resistance. Since the HMOsisEC10 had suppressed progesterone receptor (PR) expression, we transduced PR-B to HMOsisEc10 cell lines including KRAS mutant and PIK3CA mutant cell lines. We conducted a migration assay, invasion assay, and MTT assay using dienogest and medroxyprogestrone acetate. All cell lines showed progesterone sensitivity with or without mutations. Regarding inflammatory factors, real-time quantitative RT-PCR revealed that the KRAS mutation cell line exhibited no suppression of Cox-2 and mPGES-1 on progesterone treatment, whereas IL-6, MCP-1, VEGF, and CYP19A1 were significantly suppressed by progesterone in both mutated cell lines. Our results suggest that KRAS mutation and PIK3CA mutation in endometriotic cells may not be associated with progesterone resistance in terms of aggressiveness. However, KRAS mutations may be associated with progesterone resistance in the context of pain.

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

Engineered FSHD mutations results in D4Z4 heterochromatin disruption and feedforward DUX4 network activation.

Facioscapulohumeral dystrophy (FSHD) is linked to contraction of D4Z4 repeats on chromosome 4q with SMCHD1 mutations acting as a disease modifier. D4Z4 heterochromatin disruption and abnormal upregulation of the transcription factor DUX4, encoded in the D4Z4 repeat, are the hallmarks of FSHD. However, defining the precise effect of D4Z4 contraction has been difficult because D4Z4 repeats are primate-specific and DUX4 expression is very rare in highly heterogeneous patient myocytes. We generated isogenic mutant cell lines harboring D4Z4 and/or SMCHD1 mutations in a healthy human skeletal myoblast line. We found that the mutations affect D4Z4 heterochromatin differently, and that SMCHD1 mutation or disruption of DNA methylation stabilizes otherwise variegated DUX4 target activation in D4Z4 contraction mutant cells, demonstrating the critical role of modifiers. Our study revealed amplification of the DUX4 signal through downstream targets, H3.X/Y and LEUTX. Our results provide important insights into how rare DUX4 expression leads to FSHD pathogenesis.

Establishment of immortalized Egyptian Rousettus bat cell lines.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin-dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung-derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart-derived SV40 cell lines had aberrant karyotypes and the young bat-derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.

Human intestinal organoid-derived PDGFRα + mesenchymal stroma enables proliferation and maintenance of LGR4 + epithelial stem cells.

BACKGROUND: Intestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture. METHODS: We established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies. RESULTS: The proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture. CONCLUSIONS: The high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

Characterization of Common Minke Whale (Balaenoptera Acutorostrata) Cell Lines Immortalized with the Expression of Cell Cycle Regulators.

Primary cultured cells cannot proliferate infinite. The overcoming of this limit can be classified as immortalization. Bypass of p16 senescence protein induces efficient immortalization various types of mammalians is previously reported. However, the Cetacea species is not known. Here, that common minke whale-derived cells can be immortalized with a combination of human genes, mutant cyclin-dependent kinase 4 (CDK4R24C ), cyclin D1, and Telomerase Reverse Transcriptase (TERT) is reported. These results indicate that the function of cell cycle regulators in premature senescence is evolutionarily conserved. This study describes the conserved roles of cell cycle regulators in the immortalization of cells from humans to Cetacea species. Furthermore, using RNA-seq based on next-generation sequencing, the gene expression profiles of immortalized cells are compared with parental cells as well as those immortalized with SV40 large T antigen, which is once a popular method for cellular immortalization. The profiling results show that newly established common minke-whale-derived immortaliozed cells have completely different profiles from SV40 cells. This result indicates that the expression of mutant CDK4, cyclin D1, and TERT enables to establish immortalized cell lines with different biological nature from SV40 expressing cells.

Establishment and characterization of NCC-PMP2-C1: a novel patient-derived cell line of pseudomyxoma peritonei with signet ring cells.

Pseudomyxoma peritonei (PMP) is a rare phenomenon, characterized by accumulation of mucus in the abdominal cavity due to a mucinous neoplasm. Histologically, PMP is divided into three prognostic classes, namely low-grade mucinous carcinoma peritonei (LGMCP), high-grade mucinous carcinoma peritonei (HGMCP), and high-grade mucinous carcinoma peritonei with signet ring cells (HGMCP-S); HGMCP-S exhibits the worst prognosis. Complete cytoreductive surgery and hyperthermic intraperitoneal chemotherapy have been established as the standard therapy for PMP. However, 50% of patients with PMP experience a recurrence, and 30-40% are unable to receive the standard treatment due to invasive diseases. Therefore, novel therapies are required for their treatment. Although patient-derived cell lines are important tools for basic and pre-clinical research, PMP cell lines derived from patients with HGMCP-S have never been reported. Thus, we established a novel PMP cell line NCC-PMP2-C1, using surgically resected tumor tissue from a patient with HGMCP-S. NCC-PMP2-C1 cells were maintained for more than five months and passaged 30 times under culture conditions. NCC-PMP2-C1 cells exhibited multiple deletions and somatic mutations, slow growth, histological features, and dissemination of tumor cells in nude mice. Screening for the anti-proliferative effects of anti-cancer drugs on cells revealed that bortezomib, mubritinib, and romidepsin had a significant response against NCC-PMP2-C1 cells. Thus, the NCC-PMP2-C1 cell line is the first PMP cell line harboring signet ring cells and will be a valuable resource for basic and preclinical studies of HGMCP-S.

An in vitro model and the underlying pathways of sinonasal inverted papilloma development.

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.

Correction: Noguchi et al. Establishment and Characterization of NCC-PMP1-C1: A Novel Patient-Derived Cell Line of Metastatic Pseudomyxoma Peritonei. J. Pers. Med. 2022, 12, 258.

There was an error in the original publication [...].

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells.

As a metabolic organ, the liver plays a variety of roles, including detoxification. It has been difficult to obtain stable supplies of hepatocytes for transplantation and for accurate hepatotoxicity determination in drug discovery research. Human pluripotent stem cells, capable of unlimited self-renewal, may be a promising source of hepatocytes. In order to develop a stable supply of embryonic stem cell (ESC)-derived hepatocytes, we have purified human ESC-derived hepatic progenitor cells with exposure to cytocidal puromycin by using their ability to metabolize drugs. Hepatic progenitor cells stably proliferated at least 220-fold over 120 days, maintaining hepatic progenitor cell-like properties. High drug-metabolizing hepatic progenitor cells can be matured into liver cells by suppressing hepatic proliferative signals. The method we developed enables the isolation and proliferation of functional hepatic progenitors from human ESCs, thereby providing a stable supply of high-quality cell resources at high efficiency. Cells produced by this method may facilitate cell therapy for hepatic diseases and reliable drug discovery research.

Identifying high-grade serous ovarian carcinoma-specific extracellular vesicles by polyketone-coated nanowires.

Cancer cell-derived extracellular vesicles (EVs) have unique protein profiles, making them promising targets as disease biomarkers. High-grade serous ovarian carcinoma (HGSOC) is the deadly subtype of epithelial ovarian cancer, and we aimed to identify HGSOC-specific membrane proteins. Small EVs (sEVs) and medium/large EVs (m/lEVs) from cell lines or patient serum and ascites were analyzed by LC-MS/MS, revealing that both EV subtypes had unique proteomic characteristics. Multivalidation steps identified FRα, Claudin-3, and TACSTD2 as HGSOC-specific sEV proteins, but m/lEV-associated candidates were not identified. In addition, for using a simple-to-use microfluidic device for EV isolation, polyketone-coated nanowires (pNWs) were developed, which efficiently purify sEVs from biofluids. Multiplexed array assays of sEVs isolated by pNW showed specific detectability in cancer patients and predicted clinical status. In summary, the HGSOC-specific marker detection by pNW are a promising platform as clinical biomarkers, and these insights provide detailed proteomic aspects of diverse EVs in HGSOC patients.

Fusobacterium infection facilitates the development of endometriosis through the phenotypic transition of endometrial fibroblasts.

Retrograde menstruation is a widely accepted cause of endometriosis. However, not all women who experience retrograde menstruation develop endometriosis, and the mechanisms underlying these observations are not yet understood. Here, we demonstrated a pathogenic role of Fusobacterium in the formation of ovarian endometriosis. In a cohort of women, 64% of patients with endometriosis but <10% of controls were found to have Fusobacterium infiltration in the endometrium. Immunohistochemical and biochemical analyses revealed that activated transforming growth factor-ß (TGF-ß) signaling resulting from Fusobacterium infection of endometrial cells led to the transition from quiescent fibroblasts to transgelin (TAGLN)-positive myofibroblasts, which gained the ability to proliferate, adhere, and migrate in vitro. Fusobacterium inoculation in a syngeneic mouse model of endometriosis resulted in a marked increase in TAGLN-positive myofibroblasts and increased number and weight of endometriotic lesions. Furthermore, antibiotic treatment largely prevented establishment of endometriosis and reduced the number and weight of established endometriotic lesions in the mouse model. Our data support a mechanism for the pathogenesis of endometriosis via Fusobacterium infection and suggest that eradication of this bacterium could be an approach to treat endometriosis.

Optineurin deficiency impairs autophagy to cause interferon beta overproduction and increased survival of mice following viral infection.

BACKGROUND: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNß), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNß than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNß overproduction during optineurin deficiency both in vitro and in vivo. METHODS: To investigate the mechanism of IFNß overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. RESULTS: IFNß overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNß overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNß overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. CONCLUSION: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNß overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.

Sheep-derived cell immortalization through the expression of cell cycle regulators and biological characterization using transcriptomes.

Sheep are important domestic animals for the production of wool and meat. Although numerous cultured cell lines from humans and mice have been established, the number of cell lines derived from sheep is limited. To overcome this issue, the efficient establishment of a sheep-derived cell line and its biological characterization is reported. Mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase were introduced into sheep muscle-derived cells in an attempt to immortalize primary cells using the K4DT method. Furthermore, the SV40 large T oncogene was introduced into the cells. The successful immortalization of sheep muscle-derived fibroblasts was shown using the K4DT method or SV40 large T antigen. Furthermore, the expression profile of established cells showed close biological characteristics of ear-derived fibroblasts. This study provides a useful cellular resource for veterinary medicine and cell biology.

Downregulating vaccinia-related kinase 1 by luteolin suppresses ovarian cancer cell proliferation by activating the p53 signaling pathway.

OBJECTIVES: Ovarian cancer constitutes one of the most common causes of cancer-related deaths, and preventing chemotherapy resistance and recurrence in patients with ovarian cancer remains a challenge. Herein, we aimed to identify the effect of luteolin, a novel therapeutic agent targeting vaccinia-related kinase 1 (VRK1), on high-grade serous ovarian cancer (HGSOC). METHODS: Phosphokinase array, RNA sequencing, and cell cycle and apoptosis assays were conducted to determine the underlying mechanism of the effect of luteolin on HGSOC cells. The anticancer effects of oral and intraperitoneal luteolin administration were assessed in patient-derived xenograft models via several methods, including the assessment of tumor size and immunohistochemistry of phospho-p53, phosphor-HistoneH3 and cleaved caspase 3. RESULTS: Luteolin reduced HGSOC cell proliferation and increased apoptosis and cell cycle arrest at G2/M. Compared with controls, several genes were dysregulated in luteolin-treated cells, and luteolin activated the p53 signaling pathway. The human phosphokinase array revealed distinct p53 upregulation in luteolin-treated cells, as confirmed by p53 phosphorylation at ser15 and ser46 using western blot analysis. In patient-derived xenograft models, oral or intraperitoneal luteolin administration substantially suppressed tumor growth. Moreover, combination treatment involving luteolin and cisplatin inhibited tumor cell proliferation, especially in cisplatin-resistant HGSOC cell lines. CONCLUSIONS: Luteolin demonstrated considerable anticancer effect on HGSOC cells, reduced VRK1 expression, and activated the p53 signaling pathway, thereby inducing apoptosis and cell cycle arrest in G2/M and inhibiting cell proliferation. Furthermore, luteolin exhibited a synergistic effect with cisplatin both in vivo and in vitro. Thus, luteolin can be considered a promising cotreatment option for HGSOC.

Immortalization of primary cells derived from the endangered Ryukyu long-furred rat.

The Ryukyu long-furred rat is an endangered species confined to the southernmost three small islands of Japan (Amami-Oshima, Tokunoshima, and Okinawa). Its population is rapidly decreasing because of roadkill, deforestation, and feral animals. To date, its genomic and biological information are poorly understood. In this study, we successfully immortalized Ryukyu long-furred rat cells by expressing a combination of cell cycle regulators, mutant cyclin-dependent kinase 4 (CDK4R24C) and cyclin D1, together with telomerase reverse transcriptase or an oncogenic protein, the Simian Virus large T antigen. The cell cycle distribution, telomerase enzymatic activity, and karyotype of these two immortalized cell lines were analyzed. The karyotype of the former cell line immortalized with cell cycle regulators and telomerase reverse transcriptase retained the nature of the primary cells, while that of the latter cell line immortalized with the Simian Virus large T antigen had many aberrant chromosomes. These immortalized cells would be valuable for studying the genomics and biology of Ryukyu long-furred rats.

Increased lentivirus titer using ultra expression vectors.

Lentivirus is an efficient gene transfer system that is widely used in basic science. We aimed to improve viral titer by applying an ultra-expression vectors to lentiviral packaging. Application of the ultra-expression vectors increased biological titer 4 times for standard preparation. We also evaluated the efficacy of the ultra-expression vectors to relatively longer insert fragments, such as CSII-CMV-CNROE containing 5 genes in multiple cloning sites. Packaging of the ultra-expression vectors showed 3.5 times higher biological titer compared with the original method. Our improved packaging system could be applied to lentivirus to produce higher titers.

An in vitro carcinogenesis model for cervical cancer harboring episomal form of HPV16.

Deregulated expression of viral E6 and E7 genes often caused by viral genome integration of high-risk human papillomaviruses (HR-HPVs) into host DNA and additional host genetic alterations are thought to be required for the development of cervical cancer. However, approximately 15% of invasive cervical cancer specimens contain only episomal HPV genomes. In this study, we investigated the tumorigenic potential of human cervical keratinocytes harboring only the episomal form of HPV16 (HCK1T/16epi). We found that the HPV16 episomal form is sufficient for promoting cell proliferation and colony formation of parental HCK1T cells. Ectopic expression of host oncogenes, MYC and PIK3CAE545K, enhanced clonogenic growth of both early- and late-passage HCK1T/16epi cells, but conferred tumor-initiating ability only to late-passage HCK1T/16epi cells. Interestingly, the expression levels of E6 and E7 were rather lower in late-passage than in early-passage cells. Moreover, additional introduction of a constitutively active MEK1 (MEK1DD) and/or KRASG12V into HCK1T/16epi cells resulted in generation of highly potent tumor-initiating cells. Thus an in vitro model for progression of cervical neoplasia with episomal HPV16 was established. In the model, constitutively active mutation of PIK3CA, PIK3CAE545K, and overexpression of MYC, in the cells with episomal HPV16 genome were not sufficient, but an additional event such as activation of the RAS-MEK pathway was required for progression to tumorigenicity.