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.

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.

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.

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.

In vitro and in silico study of biological effects on cancer cells in the presence of metallic materials during radiotherapy.

X-ray therapy aims to eliminate tumours while minimizing side effects. Intense mucositis is sometimes induced when irradiating the oral cavity with a dental metal crown (DMC). However, the underlying mechanisms of such inducing radiosensitization by DMC remain uncertain. This study explored the radiosensitizing mechanisms around DMCs in an interdisciplinary approach with cell experiments and Monte Carlo simulation with the PHITS code. Clonogenic survival and nuclear 53BP1 foci of a cell line derived from cervical cancer cells (HeLa cells) were measured post-irradiation with therapeutic X-rays near high-Z materials such as Pb or Au plates, and the experimental sensitizer enhancement ratio (SER) was obtained. Meanwhile, the dose enhancement ratio (DER) and relative biological effectiveness for DNA damage yields were calculated using the PHITS code, by considering the corresponding experimental condition. The experiments show the experimental SER values for cell survival and 53BP1 foci near metals are 1.2-1.4, which agrees well with the calculated DER values. These suggest that the radiosensitizing effects near metal are predominantly attributed to the dose increase. In addition, as a preclinical evaluation, the spatial distributions of DER near DMC are calculated using Computed Tomography Digital Imaging and Communications in Medicine (CT-DICOM) data and a simple tooth model. As a result, the DER values evaluated using the CT-DICOM data were lower than those from a simple tooth model. These findings highlight the challenge of evaluating radiosensitizing effects near DMCs using Digital Imaging and Communications in Medicine (DICOM) images due to volume-averaging effects and emphasize the need for a high-resolution (<1 mm) dose assessment method unaffected by these effects.

Clinical investigation of use of Episil® oral solution in oral mucositis during radiotherapy for head and neck cancer.

Objective: Episil® is a bio adhesive barrier-forming oral liquid gel that has been used in recent years to relieve pain of oral mucositis (OM) with radiotherapy (RT) or chemoradiotherapy (CRT) in head and neck cancer (HNC) patients. We conducted a retrospective analysis of the clinical effects of Episil® on OM in these patients. Study design: Between June 2018 and May 2020, 65 patients with HNC were treated with RT or CRT at our hospital. Results: The median total RT dose was 50 Gy (range, 30-70 Gy) and the completion rate was 63/65 (97%). The median time to OM resolution was 47 (6-90) days and was significantly longer (53 [27-90] days) when the total RT dose was ≥51 Gy (P < 0.001). Episil® was used in 26 patients. Among them, 10 discontinued its use due to ineffective pain relief, usage difficulties, and taste intolerance. The median duration of use was 30 days and was significantly longer (34.5 days) (P < 0.001) when patients experienced pain relief at treatment initiation. Conclusion: Although Episil® has been shown to be effective in improving the pain of OM caused by RT for HNC patients, and medical professionals are required to give careful attention to each patient.

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.

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.

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.

MRI before biopsy correlates with depth of invasion corrected for shrinkage rate of the histopathological specimen in tongue carcinoma.

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.

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.

Management of retropharyngeal lymph node metastasis in oral cancer.

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.