Development of prediction models for the sensitivity of oral squamous cell carcinomas to preoperative S-1 administration.

S-1 is an anticancer agent that is comprised of tegafur, gimeracil, and oteracil potassium, and is widely used in various carcinomas including oral squamous cell carcinoma (OSCC). Although an established prediction tool is not available, we aimed to develop prediction models for the sensitivity of primary OSCC cases to the preoperative administration of S-1. We performed DNA microarray analysis of 95 cases with OSCC. Using global gene expression data and the clinical data, we developed two different prediction models, namely, model 1 that comprised the complete response (CR) + the partial response (PR) versus stable disease (SD) + progressive disease (PD), and model 2 that comprised responders versus non-responders. Twelve and 18 genes were designated as feature genes (FGs) in models 1 and 2, respectively, and, of these, six genes were common to both models. The sensitivity was 96.3%, the specificity was 91.2%, and the accuracy was 92.6% for model 1, and the sensitivity was 95.6%, the specificity was 85.2%, and the accuracy was 92.6% for model 2. These models were validated using receiver operating characteristic analysis, and the areas under the curves were 0.967 and 0.949 in models 1 and 2, respectively. The data led to the development of models that can reliably predict the sensitivity of patients with OSCC to the preoperative administration of S-1. The mechanism that regulates S-1 sensitivity remains unclear; however, the prediction models developed provide hope that further functional investigations into the FGs will lead to a greater understanding of drug resistance.

In vitro and in vivo removal of oral Candida from the denture base.

OBJECTIVES: To clarify the effectiveness of ultrasonic cleaning for removing Candida lodged in the denture base. MATERIALS AND METHODS: In vitro - Specimens of acrylic resin for denture plates contaminated with Candida cells were ultrasonically cleaned for 30 min. Washings were sampled every 5 min and cultured to investigate residual contamination, measured as colony forming units/ml, and the surfaces of the specimens were subjected to low-vacuum scanning electron microscopy (LV-SEM). In vivo - A total of 24 maxillary denture bases were brushed for 2 min under running tap water, then ultrasonically cleaned for 30 min. The washings were sampled every 5 min and cultured to investigate residual contamination. RESULTS: In vitro - Maximum removal was achieved during the first 5 min of cleaning, with the mean CFU/ml counts significantly declining over time. More than 85% of all Candida was removed within the first 15 min in specimens inoculated with phosphate-buffered saline suspensions of Candida albicans and Candida glabrata. LV-SEM revealed a decreased number of Candida on the surface of the specimens after 30 min of ultrasonic cleaning. In vivo - Maximum removal was achieved during the first 5 min of cleaning, then the mean CFU/ml count significantly declined during the first 10 min. Ultrasonic cleaning for 15 min removed 88.4% of Candida species from the denture base. CONCLUSIONS: Ultrasonic cleaning is a reliable and simple method for removing Candida lodged in the denture base.

Use of Candida-specific chicken egg yolk antibodies to inhibit the adhering of Candida to denture base materials: prevention of denture stomatitis.

OBJECTIVES: Polyclonal anti-Candida chicken egg yolk antibodies (anti-IgY) were used to investigate the prevention of adherence of Candida species to denture base material in vitro. BACKGROUND: Candida is a potential virulence factor that can cause systemic infection and even death in immunocompromised individuals. Because long-term antifungal treatment may lead to the emergence of drug-resistant strains, it is necessary to develop novel preventive measures and treatments for candidiasis. MATERIALS AND METHODS: Three types of chicken egg yolk antibodies were used in this study: non-specific antibody (control IgY), Candida albicans-specific antibody (anti-C.a.IgY) and Candida glabrata-specific antibody (anti-C.g.IgY). A mixture of different dilutions of each antibody with a suspension of Candida species and denture base material was incubated for 3 h, and then the colony-forming units of Candida on the denture base material were counted. RESULTS: Compared with control IgY, anti-C.a.IgY and anti-C.g.IgY significantly inhibited the adherence of C. albicans, but anti-C.a.IgY tended to be more potent than anti-C.g.IgY. The adherence of C. glabrata was also inhibited significantly by anti-C.a.IgY and anti-C.g.IgY with almost equivalent potency, indicating that their actions against C. glabrata were comparable. CONCLUSIONS: This study revealed the inhibitory effects of anti-C.a.IgY and anti-C.g.IgY against the adherence of C. albicans and C. glabrata to denture base material. This finding indicates the possibility of a beneficial effect of IgYs for the prevention of denture stomatitis and candidiasis in clinical settings.

Combination of MUC1 and MUC4 expression predicts clinical outcome in patients with oral squamous cell carcinoma.

BACKGROUND: Both MUC1 and MUC4 are high molecular weight glycoproteins and are independent indicators of worse prognosis in many human epithelial cancers including oral squamous cell carcinoma (OSCC). However, there has been no investigation of the clinical importance of the co-expression of MUC1 and MUC4 in OSCC. The aim of this study was to evaluate the co-expression profile of MUC1/MUC4 and analyze the prognostic significance in OSCC. METHODS: We examined the expression profile of MUC1 and MUC4 in OSCC tissues from 206 patients using immunohistochemistry. The co-expression profile of MUC1/MUC4 and its prognostic significance in OSCC was statistically analyzed. RESULTS: MUC1 and MUC4 overexpression were strongly correlated with each other (p < 0.0001) and a combination of both MUC1 and MUC4 expression was a powerful indicator for tumor aggressiveness such as tumor size (p = 0.014), lymph node metastasis (0.0001), tumor stage (p = 0.006), diffuse invasion (p = 0.028), and vascular invasion (p = 0.014). The MUC1/MUC4 double-positive patients showed the poorest overall and disease-free survival. Multivariate analysis revealed that MUC1/MUC4 double-positivity was the strong independent prognostic factor for overall and disease-free survival (p = 0.007 and (p = 0.0019), in addition to regional recurrence (p = 0.0025). CONCLUSIONS: Taken together, these observations indicate that the use of a combination of MUC1/MUC4 can predict outcomes for patients with OSCC. This combination is also a useful marker for predicting regional recurrence. MUC1 and MUC4 may be attractive targets for the selection of treatment methods in OSCC.

Frequency of clinically isolated strains of oral Candida species at Kagoshima University Hospital, Japan, and their susceptibility to antifungal drugs in 2006-2007 and 2012-2013.

BACKGROUND: The isolation frequency and susceptibility to antifungal agents of oral Candida isolates from patients with oral candidiasis (OC) were compared between studies conducted in 2006-2007 and 2012-2013. METHODS: A total158 strains was isolated from 112 patients who visited Kagoshima University Hospital for the treatment of OC during the 14-month period from February 2012 and March 2013, and evaluated on the isolation frequency of each Candida strain and the susceptibility against antifungal drugs as compared to those evaluated in 2006-2007. RESULTS: There was a higher frequency of xerostomia as a chief complaint and of autoimmune disease in the 2012-2013 study than in the 2006-2007 study. More than 95% of Candida isolates were C. albicans and C. glabrata. In addition, the proportion of the latter increased from 12.3% in the 2006-2007 study to 23.4% in the 2012-2013 study, while the proportion of the former decreased from 86.2% to 72.8%, respectively. C. albicans was isolated in almost all patients, while C. glabrata was only isolated concomitantly with C. albicans. Minimal inhibitory concentrations (MICs) were not significantly different between groups with a few exceptions. Candida isolates, of which MICs surpassed break points, apparently increased for miconazole and itraconazole against C. glabrata in the 2012-2013 study, but this was not statistically significant. As a result, more cases of autoimmune disease, a greater number of C. glabrata isolates, and higher resistance to azoles were seen in the 2012-2013 study than in the 2006-2007 study. CONCLUSION: These data indicate that with recent increases in C. glabrata infection, a causative fungus of OC, and in C. glabrata resistance to azoles, caution is needed in the selection of antifungal drugs for the treatment of OC.

Patient with oculo-facio-cardio-dental syndrome treated with surgical orthodontics.

Oculo-facio-cardio-dental (OFCD) syndrome is a rare syndrome characterized by ocular, facial, cardiac, and dental disorders. Only about 20 cases have been reported to date. The most prominent of the various features of this syndrome is canine radiculomegaly. Other features include a long and narrow face, a high nasal bridge, a broad and pointed nose, a bifid nose, ear deformity, cleft palate or submucous cleft palate, maxillary growth retardation, a large gonial angle, open apices, delayed eruption, persistent deciduous teeth, extreme overbite, and constricted maxilla. Orthodontic and prosthodontic treatment has been reported for several patients, but surgical-orthodontic treatment for OFCD has not been reported. An 18-year-old woman with skeletal Class III malocclusion and OFCD syndrome was treated with edgewise appliance therapy combined with orthognathic surgery. We applied a light force during the treatment so as not to induce ankylosis. At the end of the surgical and orthodontic treatments, functional occlusion and an improved facial profile were achieved. After the retention period, stomatognathic function was improved. The results of this treatment suggest that surgical-orthodontic treatment is an effective method for improving skeletal disharmony, facial profile, occlusion, and stomatognathic function in patients with OFCD.

DF3 epitope expression on MUC1 mucin is associated with tumor aggressiveness, subsequent lymph node metastasis, and poor prognosis in patients with oral squamous cell carcinoma.

BACKGROUND: DF3/MUC1 mucin is expressed in various cancer tissues, and many in vitro studies have suggested that it may play a role in the aggressive behavior of malignant tumors. However, to the best of the authors' knowledge, the relation between DF3/MUC1 expression and outcome has not yet been investigated in patients with oral squamous cell carcinoma (OSCC). The objective of the current study was to evaluate the prognostic significance of DF3/MUC1 expression in patients with OSCC. METHODS: The expression profile of DF3/MUC1 in OSCC tissues from 206 patients was examined using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed on the basis of detailed clinicopathologic factors. RESULTS: DF3/MUC1 expression was found to be significantly correlated with tumor aggressiveness, such as pathologic lymph node metastasis (P = .002), advanced tumor stage (P = .02), diffuse invasion of cancer cells (P = .03), and vascular invasion (P = .01). Respectively, the overall survival (OS)and disease-free survival (DFS) rates were significantly worse for patients with DF3/MUC1 expression compared with those without DF3/MUC1 expression (P = .001 and P = .0003, respectively). Multivariate analysis demonstrated that DF3/MUC1 expression was an independent prognostic factor for both OS and DFS (P = .04 for both). In addition, DF3/MUC1 expression was found to be an independent risk factor for subsequent regional lymph node metastasis (P = .03). CONCLUSIONS: Aberrant expression of DF3/MUC1 is an independent prognostic factor indicating poor prognosis in patients with OSCC. DF3/MUC1 expression is a risk factor for subsequent lymph node metastasis in patients with OSCC and therefore may represent an indication for elective neck dissection. Patients with OSCC demonstrating positive expression of DF3/MUC1 should be followed carefully.

Aberrant DNA methylation of tumor-related genes in oral rinse: a noninvasive method for detection of oral squamous cell carcinoma.

BACKGROUND: The early detection of oral squamous cell carcinoma (OSCC) is important, and a screening test with high sensitivity and specificity is urgently needed. Therefore, in this study, the authors investigated the methylation status of tumor-related genes with the objective of establishing a noninvasive method for the detection of OSCC. METHODS: Oral rinse samples were obtained from 34 patients with OSCC and from 24 healthy individuals (controls). The methylation status of 13 genes was determined by using methylation-specific polymerase chain reaction analysis and was quantified using a microchip electrophoresis system. Promoter methylation in each participant was screened by receiver operating characteristic analysis, and the utility of each gene's methylation status, alone and in combination with other genes, was evaluated as a tool for oral cancer detection. RESULTS: Eight of the 13 genes had significantly higher levels of DNA methylation in samples from patients with OSCC than in controls. The genes E-cadherin (ECAD), transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains 2 (TMEFF2), retinoic acid receptor beta (RARß), and O-6 methylguanine DNA methyltransferase (MGMT) had high sensitivity (>75%) and specificity for the detection of oral cancer. OSCC was detected with 100% sensitivity and 87.5% specificity using a combination of ECAD, TMEFF2, RARß, and MGMT and with 97.1% sensitivity and 91.7% specificity using a combination of ECAD, TMEFF2, and MGMT. CONCLUSIONS: The aberrant methylation of a combination of marker genes present in oral rinse samples was used to detect OSCC with >90% sensitivity and specificity. The detection of methylated marker genes from oral rinse samples has great potential for the noninvasive detection of OSCC.

MUC4: a novel prognostic factor of oral squamous cell carcinoma.

MUC4 mucin is now known to be expressed in various normal and cancer tissues. We have previously reported that MUC4 expression is a novel prognostic factor in several malignant tumors; however, it has not been investigated in oral squamous cell carcinoma (OSCC). The aim of our study is to evaluate the prognostic significance of MUC4 expression in OSCC. We examined the expression profile of MUC4 in OSCC tissues from 150 patients using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed. MUC4 was expressed in 61 of the 150 patients with OSCC. MUC4 expression was significantly correlated with higher T classification (p = 0.0004), positive nodal metastasis (p = 0.049), advanced tumor stage (p = 0.002), diffuse invasion of cancer cells (p = 0.004) and patient's death (p = 0.004) in OSCC. Multivariate analysis showed that MUC4 expression (p = 0.011), tumor location (p = 0.032) and diffuse invasion (p = 0.009) were statistically significant risk factors. Backward stepwise multivariate analysis demonstrated MUC4 expression (p = 0.0015) and diffuse invasion (p = 0.018) to be statistically significant independent risk factors of poor survival in OSCC. The disease-free and overall survival of patients with MUC4 expression was significantly worse than those without MUC4 expression (p < 0.0001 and p = 0.0001). In addition, the MUC4 expression was a significant risk factor for local recurrence and subsequent nodal metastasis in OSCC (p = 0.017 and p = 0.0001). We first report MUC4 overexpression is an independent factor for poor prognosis of patients with OSCC; therefore, patients with OSCC showing positive MUC4 expression should be followed up carefully.

Comprehensive epigenetic analysis using oral rinse samples: a pilot study.

PURPOSE: To prove that chromatin immunoprecipitation assay can be performed with oral rinse samples and to develop a protocol for comprehensive analysis of functional interactions among DNA methylation, histone modification, and gene expression using such samples. MATERIALS AND METHODS: Eleven cancer cell lines and oral rinse samples from 10 patients with oral squamous cell carcinoma and 3 healthy subjects were examined. The expression of CDKN2A, a tumor suppressor gene, was determined by reverse transcription/polymerase chain reaction and immunohistochemistry. Promoter DNA methylation was assessed by methylation-specific polymerase chain reaction. Chromatin modifications were analyzed by a chromatin immunoprecipitation assay using antibodies for dimethylation and acetylation of lysine 9 of histone H3. RESULTS: Epigenetic control of CDK2NA was observed in vitro in 11 cancer cell lines. Using the present protocol, comprehensive epigenetic analysis could be successfully performed with oral rinse samples. All patients were comfortable using the prescribed amount (16 mL) of normal saline to rinse their mouths. Nine patients (90%) and 1 healthy subject (33%) showed dimethylation of lysine 9 of histone H3. Moreover, 8 patients (80%) showed hypoacetylation of lysine 9 of histone H3, which was not observed in healthy subjects. CONCLUSIONS: The present study showed for the first time that chromatin modifications can be analyzed using oral rinse samples by chromatin immunoprecipitation analysis. To evaluate the contribution of histone modifications for carcinogenesis of oral squamous cell carcinoma, studies including a larger number of subjects should be conducted in the future.

Human papillomavirus in upper digestive tract tumors from three countries.

AIM: To clarify human papillomavirus (HPV) involvement in carcinogenesis of the upper digestive tract of virological and pathological analyses. METHODS: The present study examined the presence of HPV in squamous cell carcinomas of the oral cavity (n = 71), and esophagus (n = 166) collected from Japan, Pakistan and Colombia, with different HPV exposure risk and genetic backgrounds. The viral load and physical status of HPV16 and HPV16-E6 variants were examined. Comparison of p53 and p16(INK4a) expression in HPV-positive and HPV-negative cases was also made. RESULTS: HPV16 was found in 39 (55%) oral carcinomas (OCs) and 24 (14%) esophageal carcinomas (ECs). This site-specific difference in HPV detection between OCs and ECs was statistically significant (P < 0.001). There was a significant difference in the geographical distribution of HPV16-E6 variants. Multiple infections of different HPV types were found in 13 ECs, but multiple infections were not found in OCs. This difference was statistically significant (P = 0.001). The geometric means (95% confidence interval) of HPV16 viral load in OCs and ECs were 0.06 (0.02-0.18) and 0.12 (0.05-0.27) copies per cell, respectively. The expression of p16(INK4a) proteins was increased by the presence of HPV in ECs (53% and 33% in HPV-positive and -negative ECs, respectively; P = 0.036), and the high-risk type of the HPV genome was not detected in surrounding normal esophageal mucosa of HPV-positive ECs. CONCLUSION: Based on our results, we cannot deny the possibility of HPV16 involvement in the carcinogenesis of the esophagus.

Runt-related gene 2 is involved in the inhibition of matrix metalloproteinase-13 expression by roxithromycin in human gingival epithelial cell cultures.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinase (MMP)-13 has wide substrate specificity compared with other MMPs and appears to be involved in periodontitis. Previously, we reported that roxithromycin (RXM) inhibits vascular endothelial growth factor expression induced by tumour necrosis factor-alpha in human periodontal ligament cells, but little is known about the effect of RXM on MMP-13 expression in human gingival epithelial cells. We therefore examined the effect of RXM on MMP-13 mRNA expression and production in cultured human gingival epithelial cells. MATERIAL AND METHODS: Human epithelial cell lines (Ca9-22, TU4, SCCTF and HSC-3) were plated in tissue culture dishes. Then, the culture supernatants and sediments were collected and the production of MMP-13 was analysed using enzyme-linked immunosorbent assay; the expression of MMP-13 mRNA and runt-related gene 2 mRNA was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We also studied the effect of Runx2 short interfering RNA (siRNA) on the induction of MMP-13. RESULTS: Roxithromycin downregulated the induction of MMP-13 in Ca9-22 cells. Roxithromycin suppressed the expression of MMP-13 mRNA not only in Ca9-22 cells, but also in other human epithelial cell lines. Roxithromycin strongly inhibited the expression of Runx2 mRNA. Furthermore, Runx2 siRNA inhibited the induction of MMP-13 in Ca9-22 cells. CONCLUSION: These results indicate that RXM suppresses MMP-13 via the downregulation of Runx2 in human gingival epithelial cell cultures.

Orthodontic treatment combined with mandibular distraction osteogenesis and changes in stomatognathic function.

We performed an orthodontic treatment combined with mandibular distraction osteogenesis in a 15-year-old patient who wanted a correction of a chin deficiency and a protruding upper lip. The patient had an Angle Class II division 1 malocclusion with mandibular retrusion, a low mandibular plane angle, and scissors bite. First, a quad-helix appliance was applied to the mandibular dentition to correct the scissors bite in the bilateral premolar region. Later, a preadjusted edgewise appliance was applied to the maxillary and mandibular teeth. After 3 days, a mandibular distraction osteogenesis was performed. During and after the distraction, the open bite between the upper and lower dental arches was corrected using up and down elastics. The total treatment time with the edgewise appliance was 14 months. A skeletal Class I apical base relationship, good facial profile, and optimum intercuspation of the teeth were achieved with the treatment. The jaw-movement pattern on the frontal view did not change during gum chewing. However, the maximum gap without pain increased. The electromyographic (EMG) activity of the masseter and anterior temporalis muscles, and maximum occlusal force increased. The present case report suggests that an orthodontic treatment combined with mandibular distraction osteogenesis in a patient with mandibular retrusion in the late growth period might be effective for improving stomatognathic function.

Tumor lymphangiogenesis correlates with lymph node metastasis and clinicopathologic parameters in oral squamous cell carcinoma.

BACKGROUND: Lymphatic vessel density (LVD) and microvessel density (MVD) are important parameters for assessing the malignant potential of tumors and patient survival. In this report, the authors defined LVD as the density of D2-40-positive lymphatic vessels and MVD as the density of CD105-positive microvessels per unit area of tissue. It was reported previously that vascular endothelial growth factor C (VEGF-C) is a major modulator of LVD and MVD. The objectives of this study were to clarify the clinical and prognostic significance of both LVD and MVD in oral squamous cell carcinoma (OSCC) and to elucidate the lymphangiogenic and angiogenic activities of VEGF-C in cancer tissues. METHODS: In total, 110 OSCC tissue samples were evaluated for LVD, MVD, and expression of VEGF-C using immunohistochemistry. Correlations among these parameters and clinicopathologic factors were examined. RESULTS: LVD was significantly higher in tumors that had very high expression of VEGF-C compared with tumors that had no/weak expression of VEGF-C. LVD correlated well with lymph node metastasis (P < .001). MVD was correlated significantly with positive lymph node metastasis (P < .001) but not with VEGF-C expression. In contrast, high expression of VEGF-C was correlated significantly with advanced tumor status (P = .041). Survival rates were lower in patients who had higher LVD (P < .001), higher MVD (P = .0028), and strong VEGF-C expression (P = .048). CONCLUSIONS: Lymphangiogenesis predominantly influenced metastasis-free survival. The current results suggested that LVD is a more useful tool than MVD and VEGF-C for deciding on therapeutic strategies in patients with OSCC.

Hydroxyapatite formed on/in agarose gel induces activation of blood coagulation and platelets aggregation.

We reported earlier that hydroxyapatite (HA) formed on/in agarose gels (HA/agarose) produced by alternate soaking process is a bone-filling material possessing osteoconductive and hemostatic effects. This process could allow us to make bone-like apatite that was formed on/in organic polymer hydrogel matrices. Here, we investigated the mechanism of hemostasis induced by HA/agarose and found that HA/agarose, but not agarose or HA powder, significantly shortened activated partial thromboplastin time (APTT). While HA/agarose did not show significant platelet aggregation, it markedly enhanced adenosine diphosphate (ADP)-induced platelet aggregation. Moreover, Western blot analysis revealed selective adsorption of vitronectin onto HA/agarose. We also observed marked differences between HA powder and HA/agarose in their XRD patterns. The crystallinity of HA powder was much higher compared to that of HA/agarose. Furthermore, 50-100 nm of tube-form aggregations was observed in HA powder on the other hand 100-200 nm of particles was observed in HA/agarose by SEM observation. Thus 100-200 nm of low crystallized particles on the surface structure of HA/agarose may play an important role in hemostasis. Our results demonstrated a crucial role of HA/agarose in the mechanism of hemostasis and suggested a potential role for HA/agarose as a bone-grafting material.

Apatite formed on/in agarose gel as a bone-grafting material in the treatment of periodontal infrabony defect.

The present study was designed to evaluate the effects of a hydroxyapatite/agarose (HA/agarose) composite gel formed by a novel alternate soaking process for the treatment of periodontal infrabony defects in three dogs. After creating two-wall infrabony periodontal defects on the medial aspect of the maxillary and mandibular second and forth premolars, the defects were implanted with temporary dental filling material (stopping) to induce inflammatory periodontal disease. Two months later, the mucoperiosteal flaps were raised, and after debridement, the infrabony defects were filled with one of the following three materials: (a) HA/agarose, (b) Bone ject (True-Bone Ceramic-collagen combined bone graft material, Koken, Japan), or (c) no material implantation (negative control). The animals were then randomly scheduled for sacrifice at 1, 2, and 6 months, and samples were taken for histological examination. In the HA/agarose gels, the 2-month postoperative cavities exhibited regeneration to new attachments with the apposition of a new cementum and well-oriented fibers. The neocementum was narrow and acellular, and the new bone apposition was limited. Six months postoperatively, newly formed bone was predominantly observed. The neocementum was wider and cellular. In the negative control, the 2-month postoperative cavities exhibited no regeneration of the cementum, nor any formation of periodontal pockets. The six-month postoperative cavities were nearly the same as the 2-month cavities. The Bone ject, 2-month postoperative cavities exhibited no regeneration of the periodontal tissue, nor any formation of periodontal pockets. Six months postoperatively, inflammatory granulation tissue was observed around the particles. The present study suggests that HA/agarose gels may play an important role in the regeneration of lost periodontal tissue.

The assessment of cell proliferation during 9,10-dimethyl-1,2-benzanthracene-induced hamster tongue carcinogenesis by means of histone H3 mRNA in situ hybridization.

The aim of this study was to investigate cell kinetics and ultrastructural changes during carcinogenesis using a hamster 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tongue cancer model. Five squamous cell carcinomas, five dysplastic epithelia, seven hyperplastic epithelia, and four normal epithelia were obtained from 21 hamster tongues by applying 1.0% acetone solution of DMBA on the left lingual mucosa after scratching with a root canal broach. Ultrastructural examination revealed that the number of microvilli increased, whereas that of desmosomes decreased during carcinogenesis. Cell proliferation was analyzed by means of 5-bromodeoxyuridine (BrdU) immunohistochemistry and in situ hybridization (ISH) for histone H3 mRNA. The BrdU and histone H3 mRNA labeling indices (LIs) were lowest for normal epithelium, higher for hyperplastic and dysplastic epithelia, and highest for squamous cell carcinoma. Cytoplasmic histone H3 mRNA and nuclear BrdU were localized in virtually identical areas of serial sections. The correlation coefficient for the relationship between these two LIs was 0.97 ( P << 0.001). These results suggest that the assessment of cell proliferation using H3 mRNA ISH will be a useful technique for investigating biological behavior during carcinogenesis.

Expressions of junB and c-fos are enhanced in 4-nitroquinoline 1-oxide-induced rat tongue cancers.

Activator protein-1 (AP-1) is a transcription factor activated in many tumors. Using 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue cancers (TC), the present study investigated the expression levels of genes that encode the components of AP-1, the jun gene family (c-jun, junB and junD) and the fos gene family (c-fos, fra-1, fra-2 and fosB). Expression levels of junB and c-fos mRNAs in TC were significantly elevated compared with those in epithelial tissue of control rat tongue, although only c-fos mRNA levels tended to be elevated in dysplastic tongue epithelium. Histologically, all 4NQO-induced rat TC were well-differentiated squamous cell carcinomas. Immunostaining for JunB and c-Fos proteins was positive in the nuclei of tumor cells of all TC. It is noteworthy that JunB was negative, but c-Fos was positive in the dysplastic tongue epithelium of the 4NQO-treated rats. Immunostaining for both proteins was negative in tongue mucosal epithelium of control rats. There were no mutations in the coding regions of either junB or c-fos in all the TC examined. These results suggest the possibility that the expressions of junB and c-fos were enhanced stepwise in 4NQO-induced carcinogenesis of rat tongue, and that the coexpression of JunB and c-Fos might play an important role in the establishment of TC.

Osteoconductive and hemostatic properties of apatite formed on/in agarose gel as a bone-grafting material.

The biologic behavior of hydroxyapatite formed on/in agarose (HA/agarose) gels with the use of a novel alternate soaking process was compared with commercially available Bone Ject (True-Bone ceramic-collagen combined bone-graft material, Koken, Japan) as a filler for the tooth-extraction sockets of six adult monkeys (Macasa fascicularis). After the monkeys' first premolars were extracted, the defects created were replaced with one of the following materials: (a). HA/agarose created by 12 soaking cycles, (b). HA/agarose created by 9 soaking cycles, (c). Bone Ject, and (d). no material implantation (control). The time of hemostasis in each extraction site was estimated, and the samples were then studied histologically. In the controls, the time of hemostasis was about 5 min. The Bone Ject particles were easily washed out by bleeding, and the time of hemostasis was about 15 min. The HA/agarose gel was densely packed into the bony defect. The hemorrhage from the defects stopped within a few seconds after graft placement. This hemostasis was most likely related to the compactibility and adhesiveness of the material. After 12 weeks of implantation, free Bone Ject particles surrounded by inflammatory cells were observed. The bony defect filled with the HA/agarose gels was completely absorbed and replaced by newly formed bone possessing bone marrow. There was no difference in the biologic behavior of HA/agarose gels created by 9 versus 12 soaking cycles. The present study suggests that HA/agarose gels may play an important role as an alternative biodegradable bone-graft material for autogenous bone in humans.