Iqgap3-Ras axis drives stem cell proliferation in the stomach corpus during homoeostasis and repair.

OBJECTIVE: Tissue stem cells are central regulators of organ homoeostasis. We looked for a protein that is exclusively expressed and functionally involved in stem cell activity in rapidly proliferating isthmus stem cells in the stomach corpus. DESIGN: We uncovered the specific expression of Iqgap3 in proliferating isthmus stem cells through immunofluorescence and in situ hybridisation. We performed lineage tracing and transcriptomic analysis of Iqgap3 +isthmus stem cells with the Iqgap3-2A-tdTomato mouse model. Depletion of Iqgap3 revealed its functional importance in maintenance and proliferation of stem cells. We further studied Iqgap3 expression and the associated gene expression changes during tissue repair after tamoxifen-induced damage. Immunohistochemistry revealed elevated expression of Iqgap3 in proliferating regions of gastric tumours from patient samples. RESULTS: Iqgap3 is a highly specific marker of proliferating isthmus stem cells during homoeostasis. Iqgap3+isthmus stem cells give rise to major cell types of the corpus unit. Iqgap3 expression is essential for the maintenance of stem potential. The Ras pathway is a critical partner of Iqgap3 in promoting strong proliferation in isthmus stem cells. The robust induction of Iqgap3 expression following tissue damage indicates an active role for Iqgap3 in tissue regeneration. CONCLUSION: IQGAP3 is a major regulator of stomach epithelial tissue homoeostasis and repair. The upregulation of IQGAP3 in gastric cancer suggests that IQGAP3 plays an important role in cancer cell proliferation.

Involvement of IQGAP family proteins in the regulation of mammalian cell cytokinesis.

IQGAP family proteins, comprising IQGAP1, -2, and -3 in mammals, are involved in diverse ranges of cellular processes such as adhesion and migration. IQGAP proteins in yeast also play important roles in cytokinesis. However, the involvement of IQGAP proteins in cytokinesis in mammals remains unaddressed. In this study, we showed that IQGAP3 specifically localized to the equatorial cortex at anaphase, whereas IQGAP1 localized to the cell cortex uniformly and IQGAP2 was unexpressed in HeLa cells. IQGAP3, but neither IQGAP1 nor -2, was able to interact with anillin, which was required for the localization of IQGAP3 to the contractile ring. The suppressed expression of IQGAP3 inhibited the completion of cleavage furrow ingression and led to the multinucleation of cells. The suppression of IQGAP1 also had similar inhibitory effects on cytokinesis, and the simultaneous suppression of IQGAP1 and -3 induced more severe effects. The localization of anillin and RhoA to the contractile ring was impaired by the suppression of IQGAP1 and -3, whereas their upstream regulators, the centralspindlin complex and Ect2, remained unaffected. These results suggested that mammalian IQGAP proteins may play a role in cytokinesis by regulating the localization of key cytokinesis regulatory proteins to the contractile apparatus during mitosis.

A Technique for Preoperative Identification of the Facial Nerve Mandibular Branch Using a Nerve Stimulator.

We established the method of preoperative identification to facial nerve marginal mandibular branch (FNMB) identification using a nerve stimulator with bipolar probe for upper-neck surgery. The bipolar electrode is placed on the region while patients were awake; the patient should be in the same position and posture as during the surgery, with the neck skin stretched. A nerve course is confirmed by observing the movement of the lower lip. In this study, 5 upper-neck surgeries were conducted. Preoperative analysis revealed that 4 of the 5 cases had 2 branches of FNMB, and 1 with 3 branches. All FNMB immediately confirmed preoperatively were identified during surgery. We performed this method in much surgery including the surgery of the upper neck. It was easy to identify the facial nerve by this method and came to be able to do it precisely, and an operative time was shortened. We concluded that the preoperative FNMB identification using a nerve stimulator is most useful and benefit for upper-neck surgery patients and lead to avoid lower lip paralysis.

Radiation-induced fibrosarcoma after radiotherapy for osteosarcoma in the mandibular condyle.

With recent improvements in survival duration after cancer treatment, it is becoming increasingly important to study treatment-related morbidity and mortality. Radiation-induced sarcomas in the irradiated field are well-known potential late sequelae of radiotherapy. These tumors are biologically aggressive. In the case described here, a radiation-induced fibrosarcoma appeared at 20 years after initial surgery, radiotherapy, and chemotherapy for an osteosarcoma of the mandibular condyle. Radiation-induced fibrosarcoma is relatively rare in the head and neck region. The details of this case are presented, and diagnostic and management considerations are described.

ABCG2, CD44 and SOX9 are increased with the acquisition of drug resistance and involved in cancer stem cell activities in head and neck squamous cell carcinoma cells.

Cancer stem cells are a sub-population of cancer cells with self-renewal activity that play key roles in tumor resistance to chemotherapy and radiation. Several cancer stem cell markers have been identified to correlate with clinical prognosis. However, which marker is associated with which cancer stem cell characteristic is unclear. The present study aimed to clarify the relationship between cancer stem cell markers associated with drug resistance acquisition and the characteristics of cancer stem cells. We generated cisplatin-resistant head and neck squamous cell carcinoma cells by culturing cells in increasing concentrations of cisplatin. The cisplatin-resistant head and neck squamous cell carcinoma cells also acquired multidrug resistance and were named resistant HSC-3 (R HSC-3) cells. R HSC-3 showed no differences in cell proliferation or cell cycle distributions compared with parental cells. R HSC-3 cells showed increased drug excretion ability and elevated expression of ATP-binding cassette subfamily G member 2 (ABCG2), a drug excretion pump. R HSC-3 cells also highly expressed CD44, a cancer stem cell marker, and exhibited enhanced cell invasion and spheroid formation abilities. Furthermore, the stem cell-related factor SRY-box transcription factor 9 (SOX9) was identified as increased in R HSC-3 cells by microarray analysis. Knockdown experiments showed that SOX9 and ABCG2 were involved in the drug excretion ability of R HSC3 cells and ABCG2 was involved in the spheroid formation ability of R HSC-3 cells. These results indicate that CD44, SOX9 and ABCG2 expression levels were enhanced in head and neck squamous cell carcinoma cells that acquired multidrug resistance and that these molecules are important for maintaining cancer stem cell characteristics. Overall, regulating CD44, SOX9 and ABCG2 may be a strategy to inhibit cancer stem cells.

Normal establishment of epithelial tight junctions in mice and cultured cells lacking expression of ZO-3, a tight-junction MAGUK protein.

ZO-1, ZO-2, and ZO-3 are closely related MAGUK family proteins that localize at the cytoplasmic surface of tight junctions (TJs). ZO-1 and ZO-2 are expressed in both epithelia and endothelia, whereas ZO-3 is exclusively expressed in epithelia. In spite of intensive studies of these TJ MAGUKs, our knowledge of their functions in vivo, especially those of ZO-3, is still fragmentary. Here, we have generated mice, as well as F9 teratocarcinoma cell lines, that do not express ZO-3 by homologous recombination. Unexpectedly, ZO-3(-/-) mice were viable and fertile, and rigorous phenotypic analyses identified no significant abnormalities. Moreover, ZO-3-deficient F9 teratocarcinoma cells differentiated normally into visceral endoderm epithelium-like cells in the presence of retinoic acid. These cells had a normal epithelial appearance, and the molecular architecture of their TJs did not appear to be affected, except that TJ localization of ZO-2 was upregulated. Suppression of ZO-2 expression by RNA interference in ZO-3(-/-) cells, however, did not affect the architecture of TJs. Furthermore, the speed with which TJs formed after a Ca(2+) switch was indistinguishable between wild-type and ZO-3(-/-) cells. These findings indicate that ZO-3 is dispensable in vivo in terms of individual viability, epithelial differentiation, and the establishment of TJs, at least in the laboratory environment.

Human salivary gland stem/progenitor cells remain dormant even after irradiation.

Progressive loss of salivary gland function occurs in most patients undergoing head and neck radiotherapy. It is unclear whether adult salivary gland tissue contains stem/progenitor cells. In this study, we used a colony assay to clarify the presence of stem/progenitor cells in adult submandibular glands after irradiation. We developed a novel culture system that promotes single-cell colony formation with low density culture of irradiated and non-irradiated adult human submandibular gland cells using serum-free medium following serum-supplemented medium. The cells from all samples, except those obtained from the oldest patient who received the highest radiation dose, expressed acinar, ductal, and myoepithelial cell-lineage markers with reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining. A sub-culture of these colonies with serum-free medium showed high multipotency. These results are the first description of presence of salivary gland stem/progenitor cells with self-renewal, high proliferation and multipotent differentiation activity in salivary glands, even after irradiation. The survival of the cells depends on radiation dose and cell aging.

Combination with allogenic bone reduces early absorption of beta-tricalcium phosphate (beta-TCP) and enhances the role as a bone regeneration scaffold. Experimental animal study in rat mandibular bone defects.

Beta-tricalcium phosphate (beta-TCP) was grafted into rat mandibular bone defects to assess its potential as a scaffold material for bone regeneration. For this purpose, beta-TCP (TCP), allogenic bone (Allograft), and allogenic bone combined with beta-TCP (Combined) were employed as graft materials. To the left side of the graft materials in the bone defects, platelet-rich plasma (PRP) was added. The rats were sacrificed at one, three, and five weeks. Bone formation rate (BFR), remaining beta-TCP rate (RTR), beta-TCP absorption rate (TAR), whole amount of beta-TCP (WTCP), and total rate of BFR and RTR (TBR) were measured. Combined showed equivalent BFR to Allograft at five weeks, and showed higher RTR at one week and higher BFR at five weeks than TCP. Combined with PRP showed higher TAR than that without PRP at three weeks. Therefore, combination with allogenic bone showed reduced beta-TCP absorption, hence enhancing the role of beta-TCP in bone regeneration. These findings suggested that beta-TCP is a better scaffold for bone regeneration if its early absorption is reduced when used in combination with an osteogenic material.

Correction: Small-molecule inhibition of STAT3 in radioresistant head and neck squamous cell carcinoma.

[This corrects the article DOI: 10.18632/oncotarget.8368.].

Pretreatment with 5-FU enhances cisplatin cytotoxicity in head and neck squamous cell carcinoma cells.

PURPOSE: In the treatment of head and neck malignancy, cisplatin and 5-FU have been used the most as chemotherapeutic agents. The difference in efficacies of these is unclear and controversial. To investigate more effective schedule, we analyzed the cytotoxicity in different treatment sequence with two agents in vitro and the mechanism for different effectiveness. METHODS: UM-SCC-23 and UM-SCC-81B, head and neck squamous cell carcinoma cell lines, were analyzed for cellular killing in alternative sequence treatment with cisplatin and 5-FU. The treatment schedule was designed based on the clinical regimen. To determine the mechanism for the difference of cytotoxicity with each schedule, cell cycle distributions of both cells after 5-FU treatment with various durations were analyzed by flow-cytometry and immunostaining with anti-PCNA and anti-BrdU. RESULTS: 5-FU pretreatment followed by cisplatin treatment showed higher cell killing in both types of cells than the reverse treatment schedule. In the cell cycle analysis and immunostaining after the treatment of 5-FU, the rate of PCNA-positive cells was increased from 24 to 144 h in both cells. The rate of BrdU-positive cells of UM-SCC-81B in flow-cytometry was also increased, while that of UM-SCC-23 was gradually decreased. These data suggested that the cells treated with 5-FU for more than 144 h were still in the S-phase with or without DNA synthesis. CONCLUSIONS: In head and neck carcinoma cells, we showed 5-FU pretreatment enhanced cisplatin cytotoxicity. The result of cell cycle analysis and immunostaining showed S-phase arrest by treatment of prolonged 5-FU treatment. The very long arrest in S-phase might be a mechanism to enhance cisplatin cytotoxicity by 5-FU pretreatment. We thus suggest pretreatment with 5-FU to enhance the effectiveness of cisplatin-based chemotherapy.

The luminance ratio of autofluorescence in a xenograft mouse model is stable through tumor growth stages.

The aim of this research was to investigate the value of autofluorescence imaging of oral cancer across different stages of tumor growth, to assist in detecting tumors. A xenograft mouse model was created with human oral squamous cell carcinoma cell line HSC-3 being subcutaneously inoculated into nude mice. Tumor imaging was performed with an autofluorescence imaging method (Illumiscan®) using the luminance ratio, which was defined as the luminance of the tumor site over the luminance of normal skin tissue normalized to a value of 1.0. This luminance ratio was continuously observed postinoculation. Tumor and normal skin tissues were harvested, and differences in the concentrations of flavin adenine dinucleotide and nicotinamide adenine dinucleotide were examined. The luminance ratio of the tumor sites was 0.85 ± 0.05, and there was no significant change in the ratio over time, even if the tumor proliferated and expanded. Furthermore, flavin adenine dinucleotide and nicotinamide adenine dinucleotide were significantly lower in tumor tissue than in normal skin tissue. A luminance ratio under 0.90 indicates a high possibility of tumor, irrespective of the tumor growth stage. However, this cutoff value was determined using a xenograft mouse model and therefore requires further validation before being used in clinical diagnosis.

Evaluation of optimal drug concentration in histoculture drug response assay in association with clinical efficacy for head and neck cancer.

Induction chemotherapy or concomitant chemoradiotherapy has been used increasingly to improve survival, organ preservation and function in patients with head and neck cancer (HNC). However, this regimen encounters significant side effects with potential adverse reactions causing many disadvantages for patients. Therefore, reliable chemosensitivity assays are needed to accurately predict the response to chemotherapy and guide the selection and treatment of patients with HNC. The main purpose of this study was to examine the optimal drug concentrations for evaluating in vitro chemosensitivity using the histoculture drug response assay (HDRA). The tested tumor specimens included 7 from oral cavities (14.3%), 12 from oropharynx (24.5%), 10 hypopharynx (20.4%), 3 larynx (6.1%), 5 sinonasal (10.2%), 2 salivary glands (4.1%), and 10 from metastatic lymph nodes (20.4%), respectively. Histopathologic types of all 49 specimens were squamous cell carcinoma. We investigated the optimal drug concentrations in HDRA searching at doses of 4-100 microg/ml for cisplatin and 60-1500 microg/ml for 5-FU. We considered the concentration of 20 microg/ml to be appropriate for evaluating cisplatin sensitivity in HNC among the tested dosages. As for cisplatin sensitivity in vitro, the 50% cut-off inhibition index (I.I.) was found to have a significant association with the clinical response to chemotherapy, with an accurate prediction rate of 77.8%. The HDRA shows a predictive value for chemosensitivity in HNC patients using the optimal drug concentration cut-off with this site specificity.

The effect of dermatophytes on cytokine production by human keratinocytes.

Dermatophytosis (tinea) is a common disease in superficial mycoses and is generally confined to the stratum corneum in the epidermis and cutaneous appendages. The mechanisms by which dermatophytes cause dermatophytosis, however, are poorly understood. In this study, we evaluated the effect of Trichophyton mentagrophytes, T. tonsurans and T. rubrum on cytokine production by normal human epidermal keratinocytes (NHEKs). After 3-24 h of co-culture of NHEKs with each of the dermatophytes, cytokines in the supernatant were measured by enzyme-linked immunosorbent assay. Promoter activity of IL-8 was measured by chloramphenicol acetyl transferase (CAT) assay. IL-8 and GRO-alpha levels were higher in supernatants co-cultured with T. mentagrophytes isolates from animal than in those with T. mentagrophytes isolates from human, and with T. tonsurans and T. rubrum isolates. CAT expression for IL-8 promoter activity was higher in cell lysates stimulated with T. mentagrophytes isolates from animal than in those with T. mentagrophytes isolates from human, and with T. tonsurans and T. rubrum isolates. These findings suggest that dermatophytes directly induce production of cytokines at the transcriptional level by human keratinocytes, and that there are differences in their ability to induce cytokine production between the dermatophytes.

Orofacial Pain Associated with Vasospastic Angina: A Case Report.

The primary symptom of ischemic heart disease is typically chest pain, but in some cases, this pain may radiate to the maxillofacial region. This article describes the case of a 44-year-old man with orofacial pain of cardiac origin. The patient was suspected to be suffering from cardiac disease by the oral and maxillofacial surgeon and was referred to a cardiologist, where he received a heart examination. The patient was diagnosed by means of cardiac catheterization as having coronary spastic angina. During catheterization, intracoronary ergonovine maleate induced orofacial pain that was almost the same in character and intensity as the patient's first episode. The orofacial pain was considered to be telalgia from coronary spastic angina. The patient started medication on the same day as the diagnosis. There was no recurrence of any symptoms. These findings indicate that in such cases, the dentist may contribute to identifying ischemic heart disease and should refer the patient to a cardiologist.

Metastasized murine oral squamous cell carcinoma cells induce intratumoral polymorphonuclear myeloid derived suppressor cells.

Myeloid derived suppressor cells (MDSCs) localize to hematopoietic organs and peripheral blood during inflammation or tumor tissues and lymph nodes in the presence of a tumor. However, whether there are differences in MDSCs found in the primary tumor and metastases is unknown. In the present study, we established a cell line of metastasized tumor cells to a lymph node, L5-11, which were derived from the Sq-1979 mouse buccal mucosa squamous cell carcinoma cell line. We then analyzed tumor immunogenicity, especially with regard to MDSCs, to clarify the differences between the primary tumor and metastases, using an isogenic heterotopic tumor transplantation model. Our data showed that the population of intratumoral MDSCs, especially polymorphonuclear MDSCs in the lymph node metastasis model were significantly increased compared with syngeneic grafts from the primary cell line Sq-1979 after 21 days. Furthermore, we identified that the lymph node metastasis cell line had increased expression of genes that promote the expansion of MDSCs, tumor growth and metastasis. Hence, these data suggest that tumor immunosuppression can occur via activation of MDSCs. However, further examination is required to clarify whether all or a subset of these factors from the lymph node metastasis tumor cells are required to induce intratumoral MDSCs.