Differential properties of mitosis-associated events following CHK1 and WEE1 inhibitor treatments in human tongue carcinoma cells.

CHK1 and WEE1 play pivotal roles in G2/M checkpoint following exogenous DNA damage and regulation of DNA replication under normal cellular conditions. Here, we monitored and compared the cell cycle kinetics of mitosis-associated events after CHK1 and WEE1 inhibitor treatments in a human tongue cancer cell line (SAS). A fluorescent ubiquitination-based cell cycle indicator (Fucci) that reflects SCFSKP2 and APCCDH1 E3 ligase activities was used to monitor cell cycle progression. Numerous γH2AX-positive cells were observed within the S phase population of cells following CHK1 inhibitor treatment, and polyploid cells exhibiting DNA damage emerged via abortive mitosis (endomitosis) at 24 h post treatment. While WEE1 inhibitor-treated cells exhibited similar polyploidy via endomitosis at later time points, they possessed fewer γH2AX foci during S phase, and polyploid cells exhibiting DNA damage were scarce. Instead, mitosis duration greatly extended and was accompanied by an abnormal emission of Fucci red fluorescence. Kinetic analysis of Fucci fluorescence revealed that abnormal emission occurred at early M phase in a manner independent of green fluorescence degradation as a marker of APCCDH1 activation. When an inhibitor of the essential spindle checkpoint factor MPS1 was co-treated with a WEE1 inhibitor, the elongated mitosis duration and abnormal red fluorescence were abrogated, and WEE1-induced reduction of clonogenic survival was offset. We demonstrate novel differential effects on mitosis-associated events following CHK1 and WEE1 inhibitor treatments.

Regulation of p53 and Cancer Signaling by Heat Shock Protein 40/J-Domain Protein Family Members.

Heat shock proteins (HSPs) are molecular chaperones that assist diverse cellular activities including protein folding, intracellular transportation, assembly or disassembly of protein complexes, and stabilization or degradation of misfolded or aggregated proteins. HSP40, also known as J-domain proteins (JDPs), is the largest family with over fifty members and contains highly conserved J domains responsible for binding to HSP70 and stimulation of the ATPase activity as a co-chaperone. Tumor suppressor p53 (p53), the most frequently mutated gene in human cancers, is one of the proteins that functionally interact with HSP40/JDPs. The majority of p53 mutations are missense mutations, resulting in acquirement of unexpected oncogenic activities, referred to as gain of function (GOF), in addition to loss of the tumor suppressive function. Moreover, stability and levels of wild-type p53 (wtp53) and mutant p53 (mutp53) are crucial for their tumor suppressive and oncogenic activities, respectively. However, the regulatory mechanisms of wtp53 and mutp53 are not fully understood. Accumulating reports demonstrate regulation of wtp53 and mutp53 levels and/or activities by HSP40/JDPs. Here, we summarize updated knowledge related to the link of HSP40/JDPs with p53 and cancer signaling to improve our understanding of the regulation of tumor suppressive wtp53 and oncogenic mutp53 GOF activities.

Dengue Virus Type 1 Infection in Traveler Returning from Tanzania to Japan, 2019.

The largest outbreak of dengue fever in Tanzania is ongoing. Dengue virus type 1 was diagnosed in a traveler who returned from Tanzania to Japan. In phylogenetic analysis, the detected strain was close to the Singapore 2015 strain, providing a valuable clue for investigating the dengue outbreak in Tanzania.

Induction of endomitosis-like event in HeLa cells following CHK1 inhibitor treatment.

The effects of CHK1 inhibitor on cell cycle kinetics have not been fully investigated yet. In this study, we closely analyzed this kinetics using a CHK1 inhibitor (PF00477736) in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). This system allowed us to visualize cell cycle progression following CHK1 inhibitor treatment in real-time. FACS analysis showed that high levels of DNA damage as determined by γH2AX immunostaining was induced in S phase and that polyploid cells harboring the same levels of DNA damage appeared thereafter. Surprisingly, time-lapse imaging of Fucci fluorescence revealed that many cells entered M phase at once and exhibited prolonged mitosis; eventually progressing to G1 phase not accompanied by cytokinesis; this is an endomitosis-like event. Most of these cells then underwent S/G2 phases at least once, which corroborated the appearance of polyploid cells. However, a small fraction of cells with 2 N DNA content still remained 24 h after the treatment. When co-treated with MAD2 inhibitor, a core factor constituting spindle checkpoint, the 2 N DNA cell fraction disappeared and almost all cells exhibited endomitosis, leading to enhanced sensitivity. Detailed cell cycle analysis revealed that induction of an endomitosis-like event might be associated with CHK1 inhibitor-induced cell death in HeLa cells.

GII.17 norovirus infections in outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan during two decades.

Norovirus (NoV) is a major cause of viral gastroenteritis, and GII.4 has been the predominant genotype worldwide since the mid-1990s. During the 2014 to 2015 winter, a rare genotype, NoV GII.17, emerged and became prevalent mainly in East Asia. Over the past two decades, NoV molecular surveillance in Osaka City, Japan, has revealed that NoV GII.17 was detected for the first time in February 2001 and that NoV GII.17-associated outbreaks remarkably increased during the 2014 to 2015 season, with higher incidence recorded in January to March 2015. Genetic analysis indicated that 28 GII.17 outbreak strains were closely related to the novel GII.P17-GII.17 variants represented by the Kawasaki308/2015/JP strain, similar to that in other regions. Statistical analysis showed that NoV GII.17 infections were more common in adults than GII.3 and GII.4 infections, suggesting that the affected adults most likely did not have antibodies against NoV GII.17 and the novel GII.17 variant had recently appeared. Regarding transmission, food was one of the most important factors involved in the spread of NoV GII.17 among adults; 61% of GII.17 outbreaks were foodborne, with oysters being the most common vehicle. Interplay between pathogens, hosts, and environmental factors was considered to be important in the 2014 to 2015 NoV GII.17 epidemic.

Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assays for rhinovirus detection.

Human rhinoviruses (RVs) belong to the genus Enterovirus of the family Picornaviridae, and are classified into RV-A, -B, and -C species. Two assays were developed to detect RVs by a real-time fluorescent reverse transcription loop-mediated isothermal amplification method: one was designed based on the 5'-untranslated regions (UTRs) of RV-A and -B, and the other was designed based on the 5'-UTR of RV-C. The competence of both assays for the diagnosis of RV infection was tested using isolated viruses and compared with real-time reverse transcription polymerase chain reaction assays on clinical specimens. Neither assay demonstrated cross-reactivity with other tested enteroviruses, and they detected 19 out of 21 tested RV-As and seven out of eight tested RV-Cs. The specificity of the assays was 100% for the detection of RVs and their sensitivity for RV-A and RV-C was 86.3% and 77.3%, respectively, on clinical specimens by the combined use of both assays. Considering that both developed assays were highly specific and detected the majority of recently circulating RVs, they are helpful for the diagnosis of RV infection. Consequently, the results generated by these assays will enhance the surveillance of respiratory illness and the study of the roles of RVs associated with clinical features and disease severity.

Insulin-like growth factor I receptor regulates the radiation-induced G2/M checkpoint in HeLa cells.

Insulin-like growth factor I receptor (IGF-IR) plays pivotal roles in various biological events, including cell growth, transformation, survival, and DNA repair. In this study, we explored its possible involvement in cell cycle checkpoints, using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). We found that IGF-IR inhibitor delayed release from radiation-induced G2 arrest, as demonstrated by FACS and pedigree analysis of Fucci fluorescence. Elongated G2 arrest was also induced by inhibitors of phosphatidylinositol-3 kinase (PI3K) and AKT, but not by inhibitor of MEK, which are two major IGF-IR downstream signaling pathways. Double-strand break (DSB) repair kinetics were not affected by IGF-IR inhibitor. CHK1 inhibitor abrogated radiation-induced G2 arrest, whereas radiation-induced phosphorylation of CHK1 at Ser 345 or Ser 296 was decreased by the IGF-IR inhibitor. However, radiation-induced nuclear localization of CHK1 was prolonged in IGF-IR inhibitor-treated cells in comparison with cells that received radiation alone; in the latter, CHK1 returned to the original diffuse distribution in conjunction with release from G2 arrest. We conclude that IGF-IR directly regulates the G2/M checkpoint via the PI3K/AKT pathway without influencing DSB repair, in part by controlling CHK1 localization between the nucleus and cytoplasm.

Does a Vascularized Fibula Free Bone Grafted Immediately After Hemimandibulectomy in a Child Grow or Relapse During Adolescence?

For young growing children before the end of skeletal maturity, the growth activity of the grafted bone after hemimandibulectomy is not well-known. After an adolescence, such a patient may have facial deformity because the anterior growth point of the mandible is in the condylar neck. A 13-year-old boy was performed hemimandibulectomy with immediate mandibular reconstruction by fibula free flap (FFF) because of a huge ameloblastic fibroma. The authors evaluated the length of FFF on the images of computed tomography (CT) at 5 and 60 months after the operation and compared them by calculating growth rates. Five years after surgery, his facial appearance was symmetry and mandibular function was satisfaction. Although the mandibular bone in the contralateral side grew during 5-year follow-up, the vascularized FFF grafted in the child patient did not significantly grow. Moreover, spontaneous regeneration (SR) and the gradual osteosclerosis were confirmed on the left distal edge of the FFF on the CT imaging. The arrival of SR at the left distal edge of the FFF was considered a part of the reason to compensate the unchanging growth rate of the grafted FFF and contribute for the postoperative good functional and esthetic results.

Radiosensitivity of quiescent and proliferating cells grown as multicellular tumor spheroids.

The multicellular spheroid model partly mimics tumor microenvironments in vivo and has been reported in plenty of studies regarding radiosensitivity. However, clear isolation of quiescent and proliferating cells in live conditions has been quite difficult owing to technical limitations; therefore, comprehensive characterization could not be done thus far. In this study, we succeeded in separately isolating different cell types using a fluorescent ubiquitination-based cell cycle indicator (Fucci) and determining their radiosensitivities. Unexpectedly, proliferating cells were more radioresistant than quiescent cells due to the contact effect when spheroids were disaggregated immediately after irradiation. However, the radiosensitivity of quiescent cells was not influenced by mild hypoxia (hypoxia-inducible factor-1α-positive but pimonidazole-negative), but their radioresistance became similar to that of proliferating cells due to potentially lethal damage repair when disaggregated 24 h after irradiation. The Fucci system further allowed long-term observation of cell kinetics inside of the spheroid following irradiation using real-time confocal fluorescence scanning. Repeated cycles of recruitment from the quiescent to the proliferating phase resulted in cell loss from the outside of the spheroid toward the inside, causing gradual shrinkage. Interestingly, the central region of the spheroid entered a dormant stage approximately 40 days after irradiation and survived for more than 2 months. Using the Fucci system, we were able to comprehensively characterize the radiosensitivity of spheroids for the first time, which highlights the importance of cell cycle kinetics after irradiation in determining the radiosensitivity under tumor microenvironments.

ARID2 modulates DNA damage response in human hepatocellular carcinoma cells.

BACKGROUND & AIMS: Recent genomic studies have identified frequent mutations of AT-rich interactive domain 2 (ARID2) in hepatocellular carcinoma (HCC), but it is not still understood how ARID2 exhibits tumor suppressor activities. METHODS: We established the ARID2 knockout human HCC cell lines by using CRISPR/Cas9 system, and investigated the gene expression profiles and biological functions. RESULTS: Bioinformatic analysis indicated that UV-response genes were negatively regulated in the ARID2 knockout cells, and they were sensitized to UV irradiation. ARID2 depletion attenuated nucleotide excision repair (NER) of DNA damage sites introduced by exposure to UV as well as chemical compounds known as carcinogens for HCC, benzo[a]pyrene and FeCl3, since xeroderma pigmentosum complementation group G (XPG) could not accumulate without ARID2. By using large-scale public data sets, we validated that ARID2 knockout could lead to similar molecular changes between in vitro and in vivo settings. A higher number of somatic mutations in the ARID2-mutated subtypes than that in the ARID2 wild-type across various types of cancers including HCC was observed. CONCLUSIONS: We provide evidence that ARID2 knockout could contribute to disruption of NER process through inhibiting the recruitment of XPG, resulting in susceptibility to carcinogens and potential hypermutation. These findings have implications for therapeutic targets in cancers harboring ARID2 mutations. LAY SUMMARY: Recent genomic studies have identified frequent mutations of ARID2, a component of the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, in hepatocellular carcinoma, but it is not still understood how ARID2 exhibits tumor suppressor activities. In current study, we provided evidence that ARID2 knockout could contribute to disruption of DNA repair process, resulting in susceptibility to carcinogens and potential hypermutation. These findings have far-reaching implications for therapeutic targets in cancers harboring ARID2 mutations.

Impact of Coxsackievirus A6 emergence on hand, foot, and mouth disease epidemic in Osaka City, Japan.

Hand, foot, and mouth disease (HFMD) is an acute febrile illness characterized by fever; sore throat; and vesicular eruptions on the hands, feet, and oral mucosa. Until 2010, HFMD was predominantly associated with enterovirus (EV) A71 and coxsackievirus (CV) A16 in Japan. In 2011, CV-A6 emerged as a primary causative agent, causing the largest HFMD epidemic in Japan since 1981. Since then, CV-A6 has caused large HFMD epidemics every 2 years. The phylogenetic analysis of complete Viral Protein 1 (VP1) sequences revealed that most CV-A6 strains detected from 2011 to 2015 in Osaka City were classified into a different clade compared with CV-A6 strains detected from 1999 until 2009. The majority of CV-A6 strains detected in 2011 and most CV-A6 strains detected from 2013 to 2015 were mainly divided into two distinct genetic groups. Each epidemic strain carried unique amino acid substitutions in the presumed DE, EF, and GH loops of the VP1 protein that is exposed on the surface of the virion. There is a possibility that the appearance of substitutions on the surface of the virion and an accumulation of a susceptible population are significant factors in recent HFMD epidemics.

Epidemics of GI.2 sapovirus in gastroenteritis outbreaks during 2012-2013 in Osaka City, Japan.

Sapovirus (SaV) is a causative agent of gastroenteritis in humans in both sporadic cases and outbreaks. During the period from January 2005 to August 2014, SaV was detected in 30 (5.9%) of 510 gastroenteritis outbreaks in Osaka City, Japan using real-time RT-PCR. Seasonal distribution of SaV-associated outbreaks revealed an increase during the 2011-2012 season and the highest frequency of outbreaks during the 2012-2013 season. Genotyping analysis based on the capsid region demonstrated that the most common genotype was GI.2 (36.7%), in which the strains were closely related. The comparison of complete capsid gene sequences with 18 GI.2 strains (7 strains in this study and 11 from GenBank) between 1990 and 2013 showed that GI.2 strains were classified into at least three genetic clusters (1990-2000, 2004-2007, and 2008-2013) with chronologically unique amino acid residues and accumulation of mutations in the predicted P domain, suggesting the one of the causes of emergence and spread of GI.2 strains. This study will also be helpful for understanding the evolutionary mechanism of the SaV genome.

Visualizing cell-cycle kinetics after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci).

Hypoxia induces G1 arrest in many cancer cell types. Tumor cells are often exposed to hypoxia/reoxygenation, especially under acute hypoxic conditions in vivo. In this study, we investigated cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). Hypoxic treatment halted cell-cycle progression during mid-S to G2 phase, as determined by the cell cycle-regulated E3 ligase activities of SCF(Skp2) and APC/C(Cdh1), which are regulators of the Fucci probes; however, the DNA content of the arrested cells was equivalent to that in G1 phase. After reoxygenation, time-lapse imaging and DNA content analysis revealed that all cells reached G2 phase, and that Fucci fluorescence was distinctly separated into two fractions 24h after reoxygenation: red cells that released from G2 arrest after repairing DNA double-strand breaks (DSBs) exhibited higher clonogenic survival, whereas most cells that stayed green contained many DSBs and exhibited lower survival. We conclude that hypoxia disrupts coordination of DNA synthesis and E3 ligase activities associated with cell-cycle progression, and that DSB repair could greatly influence cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation.

Unusual prolongation of radiation-induced G2 arrest in tumor xenografts derived from HeLa cells.

The effect of ionizing radiation on cell cycle kinetics in solid tumors remains largely unknown because of technical limitations and these tumors' complicated structures. In this study, we analyzed intratumoral cell cycle kinetics after X-irradiation of tumor xenografts derived from HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci), a novel system to visualize cell cycle kinetics in vivo. Cell cycle kinetics after X-irradiation was examined by using tumor sections and in vivo real-time imaging system in tumor xenografts derived from HeLa cells expressing Fucci. We found that G2 arrest was remarkably prolonged, up to 5 days after 10-Gy irradiation, in contrast to monolayer cultures where G2 arrest returned within 24 h. Cells isolated from tumors 5 days after irradiation exhibited a higher surviving fraction than those isolated immediately or one day after irradiation. In this study, we clearly demonstrated unusual post-irradiation cell cycle kinetics in tumor xenografts derived from HeLa-Fucci cells. Our findings imply that prolonged G2 arrest occurring in tumor microenvironments following irradiation may function as a radioresistance mechanism.

Detection and characterization of a human G9P[4] rotavirus strain in Japan.

In a surveillance system in Osaka City, Japan, 48 sporadic rotavirus A (RVA) infections were detected during 2008/2009-2011/2012 seasons. The G/P-genotypes of detected RVAs were G1P[8], G2P[4], G3P[8], G9P[4], and G9P[8]. Although G9P[4] is a rare genotype that had not been reported in Japan, it was the second most prevalent genotype, following G1P[8], and accounted for 35.3% of RVA cases in the 2011/2012 season. Further genotyping revealed that the G9P[4] strain had genotype 2 internal protein genes except for NSP3: G9-P[4]-I2-R2-C2-M2-A2-N2-T1-E2-H2. Among detected RVA strains, G9P[4] and some G9P[8] strains shared high nucleotide identity in VP7 and NSP3 genes. Phylogenetic and BLAST search analyses showed that the G9P[4] strain in Japan shared high nucleotide identity in genotype 2 genes with common G2P[4] strains circulating globally, but was distinct from other G9P[4] strains circulating worldwide. These results suggest that the G9P[4] strain in Japan may have emerged through an independent reassortment between G9P[8] and G2P[4]. Finally, the role of NSP3 protein in the circulating RVA from an amino acid comparison between T1- and T2-type NSP3 is discussed. These findings provide an important insight into less problematic combinations of circulating RVA genes derived from different genotypes.

Gastroenteritis outbreaks caused by a DS-1-like G1P[8] rotavirus strain, Japan, 2012-2013.

Rotavirus A (RVA) genotype G1P[8], a hallmark of the Wa-like strain, typically contains only genotype 1 genes. However, an unusual RVA G1P[8] with genotype 2 genes was recently detected in Japan. We determined the complete genomic constellation of this RVA. Our findings suggest that mixed RVAs may be more competitive than once thought.

Detection and genetic characterization of human enteric viruses in oyster-associated gastroenteritis outbreaks between 2001 and 2012 in Osaka City, Japan.

Enteric viruses are an important cause of viral food-borne disease. Shellfish, especially oysters, are well recognized as a source of food-borne diseases, and oyster-associated gastroenteritis outbreaks have on occasion become international occurrences. In this study, 286 fecal specimens from 88 oyster-associated gastroenteritis outbreaks were examined for the presence of 10 human enteric viruses using antigenic or genetic detection methods in order to determine the prevalence of these infections. All virus-positive patients were over 18 years old. The most common enteric virus in outbreaks (96.6%) and fecal specimens (68.9%) was norovirus (NoV), indicating a high prevalence of NoV infection associated with the consumption of raw or under-cooked oysters. Five other enteric viruses, aichiviruses, astroviruses, sapoviruses, enteroviruses (EVs), and rotavirus A, were detected in 30.7% of outbreaks. EV strains were characterized into three rare genotypes, coxsackievirus (CV) A1, A19, and EV76. No reports of CVA19 or EV76 have been made since 1981 in the Infectious Agents Surveillance Report by the National Infectious Diseases Surveillance Center, Japan. Their detection suggested that rare types of EVs are circulating in human populations inconspicuously and one of their transmission modes could be the consumption of contaminated oysters. Rapid identification of pathogens is important for the development of means for control and prevention. The results of the present study will be useful to establish an efficient approach for the identification of viral pathogens in oyster-associated gastroenteritis in adults.

Estimating Impacts of p16 Status on Tumor Radiosensitivity in Head and Neck Cancer using Predictive Models.

The intrinsic radiosensitivity index (RSI) and genomic-adjusted radiation dose (GARD) were reported to be able to predict the surviving fraction at 2 Gy and therapeutic effect when delivering actual treatment doses using the gene expression profiles of clinical cases. Given the impact of p16 status, a surrogate marker of the human papillomavirus (HPV) infection, on radiosensitivity, we attempted to apply the RSI and GARD to estimate p16-associated radiosensitivity in head and neck squamous cell carcinoma (HNSC). For this purpose, The Cancer Genome Atlas (TCGA) dataset was employed. In the GARD calculation, we assumed that p16-positive patients received 60 Gy in 30 fractions, while p16-negative patients received 70 Gy in 35 fractions. p16 positivity was associated with favorable characteristics compared to negative patients. The RSI and GARD analyses demonstrated increased radiosensitivity and high therapeutic effect in p16-positive patients, compared to p16-negative patients. Additionally, tumor microenvironmental conditions predicted by other models were also significantly affected by p16 status. Collectively, the models used in this study could be a promising tool for estimating p16-associated radiosensitivity in HNSC.

Prevalence of human adenovirus type 3 associated with pharyngoconjunctival fever in children in Osaka, Japan during and after the COVID-19 pandemic.

Since the COVID-19 pandemic has affected the epidemiological pattern of pharyngoconjunctival fever (PCF) caused by human adenovirus (HAdV), the prevalence and type distribution of HAdVs associated with PCF among children in Osaka, Japan, between 2019 and 2023 have been analyzed. The number of reported PCF cases in Osaka decreased from 2020 to 2022, followed by an unprecedented increase in 2023. HAdV-C strains, including types C1, C2, and C5, were annually detected in pathogen surveillance in Osaka. HAdV-B3 was not detected for 2 years and 9 months from March 2020, and the number of detections increased from July 2023. In total, HAdV-B3 was the most frequently detected (27 of 52 strains), and genetic analysis of its hexon hypervariable regions showed that, except for one strain, the HAdV-B3 strains identified after 2022 had different amino acid substitutions compared to those identified in 2019 and 2020. These results suggest that the PCF epidemic in 2023 was predominantly caused by variant strains of HAdV-B3, and children who have not acquired immunity against HAdV-B3 between 2020 and 2022 were thought to be infected. The impact of COVID-19 on the prevalence of HAdV infections needs to be continuously evaluated through surveillance.

DNA damage response in a 2D-culture model by diffusing alpha-emitters radiation therapy (Alpha-DaRT).

Diffusing alpha-emitters radiation therapy (Alpha-DaRT) is a unique method, in which interstitial sources carrying 224Ra release a chain of short-lived daughter atoms from their surface. Although DNA damage response (DDR) is crucial to inducing cell death after irradiation, how the DDR occurs during Alpha-DaRT treatment has not yet been explored. In this study, we temporo-spatially characterized DDR such as kinetics of DNA double-strand breaks (DSBs) and cell cycle, in two-dimensional (2D) culture conditions qualitatively mimicking Alpha-DaRT treatments, by employing HeLa cells expressing the Fucci cell cycle-visualizing system. The distribution of the alpha-particle pits detected by a plastic nuclear track detector, CR-39, strongly correlated with γH2AX staining, a marker of DSBs, around the 224Ra source, but the area of G2 arrested cells was more widely spread 24 h from the start of the exposure. Thereafter, close time-lapse observation revealed varying cell cycle kinetics, depending on the distance from the source. A medium containing daughter nuclides prepared from 224Ra sources allowed us to estimate the radiation dose after 24 h of exposure, and determine surviving fractions. The present experimental model revealed for the first time temporo-spatial information of DDR occurring around the source in its early stages.