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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. [ABSTRACT FROM AUTHOR]

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. • We studied CHK1 inhibitor-induced cell cycle kinetics in HeLa-Fucci cells. • DNA damage was induced in S phase and mitotic duration was elongated. • CHK1 inhibitor induced endomitosis and polyploidy. • MAD2 inhibitor co-treatment enhanced endomitosis and reduced cell survival. • Endomitosis may be linked to CHK1-induced cell death in HeLa cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate which radiological depth of invasion (r-DOI) measurement is the most concordant to clinical DOI (c-DOI) derived from correction for the shrinkage rate of the histopathological specimens. We retrospectively reviewed 128 patients with tongue carcinoma who had undergone glossectomy between 2006 and 2019. At first, the width shrinkage rate during formalin fixation and preparation process of histopathological specimens was evaluated. From the shrinking rates, a formula to calculate c-DOI from pathological DOI (p-DOI) was developed. The correlation between c-DOI and r-DOI was evaluated. The specimen shrinkage rate during the histopathological specimen preparation process was 10.3%. Based on that, we yielded the correct formula for c-DOI based on p-DOI and preparation shrinkage rate: c-DOI = p-DOI × 100/89.7. The regression equations for the association of c-DOI with r-DOI measured by ultrasound (n = 128), MRI before biopsy (n = 18), and MRI after biopsy (n = 110) were y = 1.12 * x + 0.21, y = 0.89 * x − 0.26, and y = 0.52 * x + 2.63, respectively, while the coefficients of determination were 0.664, 0.891, and 0.422, respectively. In conclusion, r-DOI using MRI before biopsy most strongly correlated with c-DOI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objectives: Retropharyngeal lymph node (RPLN) metastasis is extremely rare, and prognosis is significantly poor in oral cancer. We retrospectively examined the management of RPLN metastases in oral cancer.Materials and Methods: A total of 1247 patients with oral cancer were treated at our department from January 2002 and December 2016. Among these patients, 374 (30%) had histologically positive lymph node metastases. Of these, 15 patients (1.2%) were diagnosed with RPLN metastases. We evaluated the diagnostic period, size, recurrence pattern, laterality, treatment, and therapeutic outcomes. The Kaplan-Meier method was used to determine overall survival (OS) among the RPLN metastasis group, cervical lymph node (CLN) metastases group, and treatment methods group for RPLN metastases.Results: One patient had RPLN involvement at the initial treatment, and RPLN involvement in other patients was found subsequently. The mean duration in confirming RPLN metastases was 228 days (range, 50-867 days). Surgical therapy was performed in 5 patients, chemoradiotherapy in 7 patients, and best supported care (BSC) in 3 patients. The cumulative 5-year OS rate for the RPLN metastasis group (n = 15) was 38.1%, compared with the rate of 71.3% for the CLN group (n = 359). Regarding the therapeutic approach for RPLN metastases, OS rates were 80.0% (n = 5) in the surgical therapy group, 28.6% (n = 7) in the chemoradiotherapy group, and 0% (n = 3) in the BSC group.Conclusion: Early detection and surgical treatment of RPLN metastases are associated with increased survival rate in oral cancer. [ABSTRACT FROM AUTHOR]