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.

Retrospective analysis of odontogenic myxoma and odontogenic myxofibroma in the oral and maxillofacial region: a fibrous tissue-related differentiation.

The clinical differences between odontogenic myxoma (OM) and odontogenic myxofibroma (OMF), and the clinical significance of their classifications, remain unclear. This study reviewed the clinicopathological characteristics of patients with OM or OMF and evaluated the fibrous component of the specimens. Medical records of 21 patients with OM or OMF who underwent tumour resection were reviewed. The percentage of fibrous tissue on the representative sections was evaluated using haematoxylin and eosin- and Masson's trichrome-stained specimens. Histopathological diagnoses included 11 OMs and 10 OMFs with no tumour recurrence except for two cases in which the dredging method was applied. More cortical bone perforation was observed in OM than in OMF cases, without significant differences. Location-locularity and apparent diffusion coefficient value (ADC)-cortical bone perforation were significantly correlated in all OM and OMF cases. The percentage of fibrous tissue in specimens showed bimodal distribution bordered by 45%. There was a significant association between diagnosis based on 45% fibrous tissue criterion and the final pathological diagnosis. Our study showed a tendency for cortical bone perforation in OM compared to OMF and correlation between ADC and cortical bone perforation. According to the histopathological analyses, the fibrous component of each case was bimodal with 45%, which may be a criterion to distinguish between OM and OMF. Accumulating knowledge, such as significant differences in prognosis, may allow for minimal surgical treatment options based on the diagnosis according to this novel histopathological criterion.

Clinicopathological analysis of 134 patients with squamous cell carcinoma of the mandibular gingiva.

Objective: The accurate assessment of the involvement of mandibular gingival squamous cell carcinoma (SCC) is essential for determining the extent of resection and is also useful for predicting lymph node metastasis and prognosis. The purpose of this study was to investigate the factors for predicting the prognosis. Study design: We reviewed 134 patients with mandibular gingival SCC treated between 2008 and 2017. The clinical findings, TN stage, relationship between radiographical type and histological pattern, and factors affecting the survival rate were investigated. Results: The moth-eaten radiographic type was significantly associated with histologically infiltrative pattern. For all 134 cases, the 5-year OS was 89.5 %, and 5-year DSS was 93.9 %. The 5-year DSS was 95.0 % for cN0 and/or pN0 cases and 90.3 % for pN (+) cases, with a significant difference. The significant risk factors for lymph node metastasis were teeth extractions by previous physicians and moth-eaten radiographic type. Conclusion: The risk factor for poor prognosis was lymph node metastasis. In addition, teeth extractions by previous physicians and moth-eaten radiographic type were the risk factors for lymph node metastasis. It is recommended that these cases be treated considering the possibility of cervical lymph node metastasis.

Characterization of an Mtbp Hypomorphic Allele in a Diethylnitrosamine-Induced Liver Carcinogenesis Model.

MTBP is implicated in cell cycle progression, DNA replication, and cancer metastasis. However, the function of MTBP remains enigmatic and is dependent on cellular contexts and its cellular localization. To understand the in vivo physiological role of MTBP, it is important to generate Mtbp knockout mice. However, complete deletion of the Mtbp gene in mice results in early embryonic lethality, while its heterozygous deletion shows modest biological phenotypes, including enhanced cancer metastasis. To overcome this and better characterize the in vivo physiological function of MTBP, we, for the first time, generated mice that carry an Mtbp hypomorphic allele (MtbpH) in which Mtbp protein is expressed at approximately 30% of that in the wild-type allele. We treated wild-type, Mtbp+/-, and MtbpH/- mice with a liver carcinogen, diethylnitrosamine (DEN), and found that the MtbpH/- mice showed worse overall survival when compared to the wild-type mice. Consistent with previous reports using human liver cancer cells, mouse embryonic fibroblasts (MEFs) from the MtbpH/- mice showed an increase in the nuclear localization of p-Erk1/2 and migratory potential. Thus, MtbpH/- mice and cells from MtbpH/- mice are valuable to understand the in vivo physiological role of Mtbp and validate the diverse functions of MTBP that have been observed in human cells.

Shedding of rubella virus in postsymptomatic individuals; viral RNA load is a potential indicator to estimate candidate patients excreting infectious rubella virus.

BACKGROUND: Since the first isolation of rubella virus (RuV) in 1962, comprehensive data regarding the quantitative evaluation of RuV shedding remain unavailable. In this study, we evaluated the shedding of viral RNA and infectious virus in patients with acute RuV infection. STUDY DESIGN: We analyzed 767 specimens, including serum/plasma, peripheral blood mononuclear cells (PBMCs), throat swabs, and urine, obtained from 251 patients with rubella. The viral RNA load and the presence of infectious RuV were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and virus isolation. RESULTS: Virus excretion peaked 0-2 days after rash onset and decreased over time. The median viral RNA load dropped to an undetectable level on day 3 after rash onset in serum/plasma, day 2 in PBMCs, days 10-13 in throat swabs, and days 6-7 in urine. Infectious virus could be isolated for up to day 2 after rash onset in serum/plasma, day 1 in PBMCs, days 8-9 in throat swabs, and days 4-5 in urine. The minimum viral RNA load that allowed virus isolation was 961 copies/mL in serum/plasma, 784 copies/mL in PBMCs, 650 copies/mL in throat swabs, and 304 copies/mL in urine. A higher viral RNA load indicated a higher likelihood of the presence of infectious virus. CONCLUSION: These findings would contribute to improve algorithms for rubella surveillance and diagnosis. In addition, this study indicates that the results of RT-qPCR enable efficient rubella control by estimating candidate patients excreting infectious virus, which could help prevent viral transmission at an early stage and eliminate rubella ultimately.

Retrograde Migration of an Au-198 Grain to the Submandibular Gland Post Brachytherapy Treatment of Floor of Mouth Cancer.

At our institution, radiation oncologists routinely treat early-stage oral cancer with low-dose-rate brachytherapy (LDR-BRT) using Au-198 grains. In this report, we show a unique case of a patient with a gold grain located within the submandibular gland, found incidentally during follow-up after LDR-BRT for floor of mouth cancer. One month after the implant, he showed sialadenitis-like symptoms, but the pain resolved two months later. All the grains were detected around the anterior sublingual area by computed tomography (CT) four months after the implant. Unexpectedly, 11 months after the implant, CT revealed that a grain was located in an intraglandular site of the submandibular gland. This finding clearly demonstrates that the grain entered Wharton's duct and retrogradely migrated to the submandibular gland through the duct. As a mechanism of the calculus formation within Wharton's duct, retrograde migration of foreign bodies to the inside of the duct has been proposed. Our incidental finding after LDR-BRT highlights the need for monitoring post-LDR-BRT using Au-198 grains for the treatment of floor of mouth cancer and sheds additional light on retrograde theory within Wharton's duct.

DNAJA1- and conformational mutant p53-dependent inhibition of cancer cell migration by a novel compound identified through a virtual screen.

Cancers are frequently addicted to oncogenic missense mutant p53 (mutp53). DNAJA1, a member of heat shock protein 40 (HSP40), also known as J-domain proteins (JDPs), plays a crucial role in the stabilization and oncogenic activity of misfolded or conformational mutp53 by binding to and preventing mutp53 from proteasomal degradation. However, strategies to deplete mutp53 are not well-established, and no HSP40/JDPs inhibitors are clinically available. To identify compounds that bind to DNAJA1 and induce mutp53 degradation, we performed an in silico docking study of ~10 million of compounds from the ZINC database for the J-domain of DNAJA1. A compound 7-3 was identified, and its analogue A11 effectively reduced the levels of DNAJA1 and conformational mutp53 with minimal effects on the levels of wild-type p53 and DNA-contact mutp53. A11 suppressed migration and filopodia formation in a manner dependent on DNAJA1 and conformational mutp53. A mutant DNAJA1 with alanine mutations at predicted amino acids (tyrosine 7, lysine 44, and glutamine 47) failed to bind to A11. Cells expressing the mutant DNAJA1 became insensitive to A11-mediated depletion of DNAJA1 and mutp53 as well as A11-mediated inhibition of cell migration. Thus, A11 is the first HSP40/JDP inhibitor that has not been previously characterized for depleting DNAJA1 and subsequently conformational mutp53, leading to inhibition of cancer cell migration. A11 can be exploited for a novel treatment against cancers expressing conformational mutp53.

Determination of Clonogenic Radiosensitivity of HeLa Cells in Early and Late G1 Phases.

BACKGROUND/AIM: Using a fluorescent ubiquitination-based cell cycle indicator (Fucci), we recently reported that post irradiation of HeLa cells, micronuclei frequency increased in the early G1 phase in comparison with that in the late G1 phase. This is inconsistent with the results of well-recognized studies that used clonogenic assays. In this study, we determined radiosensitivity of the cells using a clonogenic assay by making the best use of the Fucci property, while simultaneously characterizing cell cycle kinetics and DNA damage responses. MATERIALS AND METHODS: Early and late G1 phase cell fractions were isolated using a cell sorter by exploiting the different red fluorescence intensities of Fucci. Radiosensitivity was determined by the colony formation assay. Time-lapse imaging and immunostaining were performed to analyze cell cycle kinetics and DNA damage. RESULTS: Late G1 cells were more radioresistant than early G1 cells. Cells irradiated in the early and late G1 phases induced G2 arrest, while the latter demonstrated a significantly longer duration of G2 arrest. This difference became more evident as the radiation dose increased. Furthermore, 16 h after irradiation, a greater number of γH2AX foci remained in cells irradiated in the early G1 phase than in those irradiated in the late G1 phase. CONCLUSION: HeLa cells in the late G1 phase are more radioresistant than those in the early G1 phase, presumably because DNA damage is efficiently repaired during a longer G2 arrest in late G1 cells.

Effect of Ablative Dose Irradiation on Redistribution and Radioresponse in a Mouse Xenograft Model.

We investigated the effects of ablative dose irradiation on redistribution and radioresponse after the second irradiation in a mouse xenograft model, assuming stereotactic body radiotherapy (SBRT). A human tongue cancer cell line, SAS-Fucci, expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci) that visualizes the cell cycle, was employed in this study. Tumor xenografts formed subcutaneously in nude mice (approximately 6 mm in diameter), with essentially no hypoxic regions, were irradiated at 10 Gy and G2 arrest kinetics were determined using histology sections and a real-time detection method. The second irradiation (10 Gy) was given at intervals of 0 h, 3 h, 1 day, and 4 days after the first irradiation, and tumor regrowth curves were obtained. It was revealed that the ratio of G2-arrested cells showed a much higher peak at 1 day postirradiation compared to 2 Gy, assuming conventional radiotherapy, and gradually decreased thereafter up to 4 days. Tumors irradiated at intervals of 0 h and 1 day demonstrated significantly higher radioresponses than other timings. We conclude that redistribution could contribute to the efficacy of SBRT.

Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin.

Accumulation of missense mutant p53 (mutp53) in cancers promotes malignant progression. DNAJA1, a member of HSP40 (also known as J-domain proteins: JDPs), is shown to prevent misfolded or conformational mutp53 from proteasomal degradation. Given frequent addiction of cancers to oncogenic mutp53, depleting mutp53 by DNAJA1 inhibition is a promising approach for cancer therapy. However, there is no clinically available inhibitor for DNAJA1. Our in silico molecular docking study with a natural compound-derived small molecule library identified a plumbagin derivative, PLIHZ (plumbagin-isoniazid analog), as a potential compound binding to the J domain of DNAJA1. PLIHZ efficiently reduced the levels of DNAJA1 and several conformational mutp53 with minimal impact on DNA contact mutp53 and wild-type p53 (wtp53). An analog, called PLTFBH, which showed a similar activity to PLIHZ in reducing DNAJA1 and mutp53 levels, inhibited migration of cancer cells specifically carrying conformational mutp53, but not DNA contact mutp53, p53 null, and wtp53, which was attenuated by depletion of DNAJA1 or mutp53. Moreover, PLTFBH reduced levels of multiple other HSP40/JDPs with tyrosine 7 (Y7) and/or tyrosine 8 (Y8) but failed to deplete DNAJA1 mutants with alanine substitution of these amino acids. Our study suggests PLTFBH as a potential inhibitor for multiple HSP40/JDPs.

TGF-ß generates a population of cancer cells residing in G1 phase with high motility and metastatic potential via KRTAP2-3.

Transforming growth factor ß (TGF-ß) increases epithelial cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). TGF-ß also inhibits cell proliferation by inducing G1 phase cell-cycle arrest. However, the correlation between these tumor-promoting and -suppressing effects remains unclear. Here, we show that TGF-ß confers higher motility and metastatic ability to oral cancer cells in G1 phase. Mechanistically, keratin-associated protein 2-3 (KRTAP2-3) is a regulator of these dual effects of TGF-ß, and its expression is correlated with tumor progression in patients with head and neck cancer and migratory and metastatic potentials of oral cancer cells. Furthermore, single-cell RNA sequencing reveals that TGF-ß generates two populations of mesenchymal cancer cells with differential cell-cycle status through two distinctive EMT pathways mediated by Slug/HMGA2 and KRTAP2-3. Thus, TGF-ß-induced KRTAP2-3 orchestrates cancer cell proliferation and migration by inducing EMT, suggesting motile cancer cells arrested in G1 phase as a target to suppress metastasis.

Roles of IGFBP-3 in cell migration and growth in an endophytic tongue squamous cell carcinoma cell line.

Insulin-like growth factor binding protein-3 (IGFBP-3) is a member of the IGFBP family that has high affinity for IGFs and functions as either an oncogene or tumor suppressor in various types of cancer. We previously found that IGFBP3 mRNA levels are higher in endophytic-type human tongue squamous cell carcinoma (TSCC) that is more invasive and more prone to metastasis than exophytic and superficial types. This finding prompted us to investigate the roles of IGFBP-3 in TSCC using SAS cells, which were originally derived from endophytic-type TSCC. Specifically, we used SAS cells that express a fluorescent ubiquitination-based cell-cycle indicator (Fucci). RNA-sequencing analysis indicated that IGFBP-3 is associated with cell migration and cell growth. In fact, IGFBP-3 knockdown downregulates cell migration and causes cells to arrest in G1. This migratory potential appears to be cell cycle-independent. IGFBP-3 knockdown also reduced levels of secreted IGFBP-3; however, decreased migratory potential was not rescued by exogenous recombinant human IGFBP-3. Furthermore, ERK activity was downregulated by IGFBP-3 depletion, which suggests that MEK/ERK signaling may be involved in IGFBP-3-mediated cell migration. We therefore conclude that intracellular IGFBP-3 enhances cell migration independently of the cell cycle in TSCC with a higher metastatic potential.

Differences in the stemness characteristics and molecular markers of distinct human oral tissue neural crest-derived multilineage cells.

OBJECTIVES: Although multilineage cells derived from oral tissues, especially the dental pulp, apical papilla, periodontal ligament, and oral mucosa, have neural crest-derived stem cell (NCSC)-like properties, the differences in the characteristics of these progenitor cell compartments remain unknown. The current study aimed to elucidate these differences. MATERIAL AND METHODS: Sphere-forming apical papilla-derived cells (APDCs), periodontal ligament-derived cells (PDLDCs), and oral mucosa stroma-derived cells (OMSDCs) from the same individuals were isolated from impacted developing teeth. All sphere-forming cells were characterized through biological analyses of stem cells. RESULTS: All sphere-forming cells expressed neural crest-related markers. The expression of certain tissue-specific markers such as CD24 and CD56 (NCAM1) differed among tissue-derived cells. Surprisingly, the expression of only CD24 and CD56 could be discriminated in human tissues. Although APDCs and PDLDCs exhibited greater mineralized cell differentiation than OMSDCs, they exhibited poorer differentiation into adipocytes in vitro. In immunocompromised mice, APDCs formed hard tissues better than PDLDCs and OMSDCs. CONCLUSIONS: Although cells with NCSC-like properties present the same phenotype, they differ in the expression of certain markers and differentiation abilities. This study is the first to demonstrate the differences in the differentiation ability and molecular markers among multilineage human APDCs, PDLDCs, and OMSDCs obtained from the same patients, and to identify tissue-specific markers that distinguish tissues in the developing stage of the human tooth with immature apex.

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.

Efficacy and Safety of Induction Chemotherapy and/or External Beam Radiotherapy Followed by Brachytherapy in Patients With Tongue Cancer.

BACKGROUND/AIM: To evaluate the outcomes of induction chemotherapy and/or external beam radiotherapy (EBRT) followed by brachytherapy (BT) in patients with tongue cancer who had a waiting period for BT or a large tumor that was not suitable for BT. PATIENTS AND METHODS: As an induction therapy, chemotherapy with tegafur/gimeracil/oteracil (S-1), EBRT or both S-1 chemotherapy and EBRT was prescribed. BT was performed using Au-198 grains or Ir-192 pins. Local control (LC), lymph node metastasis-free survival (LNMFS), overall survival (OS), and complication rates were calculated. RESULTS: Forty-nine patients were enrolled in this study. The 3-year LC, LNMFS, and OS rates for cT1-2 patients were 84%, 45%, and 69%, respectively. The 3-year LC, LNMFS, and OS rates for cT3 patients were 77%, 58%, and 79%, respectively. The incidence of Grade 3 or 4 complications was 6%. CONCLUSION: Induction therapy contributed to the efficacy of the subsequent BT in LC rate.

DNAJA1 promotes cancer metastasis through interaction with mutant p53.

Accumulation of mutant p53 (mutp53) is crucial for its oncogenic gain of function activity. DNAJA1, a member of J-domain containing proteins or heat shock protein 40, is shown to prevent unfolded mutp53 from proteasomal degradation. However, the biological function of DNAJA1 remains largely unknown. Here we show that DNAJA1 promotes tumor metastasis by accumulating unfolded mutp53. Levels of DNAJA1 in head and neck squamous cell carcinoma (HNSCC) tissues were higher than those in normal tissues. Knockdown of DNAJA1 in HNSCC cell lines carrying unfolded mutp53 significantly decreased the levels of mutp53, filopodia/lamellipodia formation, migratory potential, and active forms of CDC42/RAC1, which were not observed in HNSCC cells with DNA contact mutp53, wild-type p53, or p53 null. Such mutp53-dependent functions of DNAJA1 were supported by the observation that DNAJA1 selectively bound to unfolded mutp53. Moreover, DNAJA1 knockdown in HNSCC cells carrying unfolded mutp53 inhibited primary tumor growth and metastases to the lymph nodes and lungs. Our study suggests that DNAJA1 promotes HNSCC metastasis mainly in a manner dependent on mutp53 status, suggesting DNAJA1 as a potential therapeutic target for HNSCC harboring unfolded mutp53.

Visualization of Radiation-Induced Cell Cycle Kinetics with a Fluorescent Ubiquitination-Based Cell Cycle Indicator (Fucci).

Among the methods for detecting cell cycle kinetics in tumor cells, fluorescent ubiquitination-based cell cycle indicator (Fucci) is innovative because it allows observation in live cells without losing spatiotemporal information. We succeeded in using the Fucci system to visualize radiation-induced G2 arrest in tumor cells with deficient p53 function. Here we describe protocols for establishing Fucci-expressing cell lines and analyzing radiation-induced G2 arrest kinetics in three different models: monolayer cell cultures, spheroids, and xenografted solid tumors in mice.

Prolonged cetuximab treatment promotes p27Kip1-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma.

Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is an efficient anti-tumor therapeutic agent that inhibits the activation of EGFR; however, data related to the cellular effects of prolonged cetuximab treatment are limited. In this study, the long-term cellular outcome of prolonged cetuximab treatment and the related molecular mechanism were explored in a head and neck squamous cell carcinoma cell line constitutively expressing a fluorescent ubiquitination-based cell cycle indicator. Fluorescent time-lapse imaging was used to assess clonal growth, cell motility, and cell-cycle progression. Western blot analysis was performed to measure the level of phosphorylation and protein-expression following cetuximab treatment. Over 5 days cetuximab treatment decreased cell motility and enhanced G1 phase cell arrest in the central region of the colonies. Significantly decreased phosphorylation of retinoblastoma, Skp2, and Akt-mTOR proteins, accumulation of p27Kip1, and induction of type II LC3B were observed over 8 days cetuximab treatment. Results of the present study elucidate the cetuximab-dependent inhibition of cell migration, resulting in high cell density-related stress and persistent cell-cycle arrest at G1 phase culminating in autophagy. These findings provide novel molecular insights related to the anti-tumor effects of prolonged cetuximab treatment with the potential to improve future therapeutic strategy.

Fluctuation in radioresponse of HeLa cells during the cell cycle evaluated based on micronucleus frequency.

In this study, we examined the fluctuation in radioresponse of HeLa cells during the cell cycle. For this purpose, we used HeLa cells expressing two types of fluorescent ubiquitination-based cell cycle indicators (Fucci), HeLa-Fucci (CA)2 and HeLa-Fucci (SA), and combined this approach with the micronucleus (MN) assay to assess radioresponse. The Fucci system distinguishes cell cycle phases based on the colour of fluorescence and cell morphology under live conditions. Time-lapse imaging allowed us to further identify sub-positions within the G1 and S phases at the time of irradiation by two independent means, and to quantitate the number of MNs by following each cell through M phase until the next G1 phase. Notably, we found that radioresponse was low in late G1 phase, but rapidly increased in early S phase. It then decreased until late S phase and increased in G2 phase. For the first time, we demonstrated the unique fluctuation of radioresponse by the MN assay during the cell cycle in HeLa cells. We discuss the difference between previous clonogenic experiments using M phase-synchronised cell populations and ours, as well as the clinical implications of the present findings.

Publisher Correction: Clinical evaluation of fully automated molecular diagnostic system "Simprova" for influenza virus, respiratory syncytial virus, and human metapneumovirus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.