Transcriptomic analysis of Porphyromonas gingivalis-infected head and neck cancer cells: Identification of PLAU as a candidate prognostic biomarker.

Periodontal disease is a risk factor for head and neck squamous cell carcinoma (HNSCC), and Porphyromonas gingivalis, a major periodontal pathogen, has been identified as a specific and potentially independent microbial factor that increases the risk of cancer mortality. Gene expression in HNSCC due to P. gingivalis infection and how changes in gene expression affect the prognosis of HNSCC patients are not clarified. When P. gingivalis was cultured with HNSCC cells, it efficiently adhered to these cells and enhanced their invasive ability. A transcriptome analysis of P. gingivalis -infected HNSCC cells showed that genes related to migration, including CCL20, CITED2, CTGF, C8orf44-SGK3, DUSP10, EGR3, FUZ, HBEGF, IL1B, IL24, JUN, PLAU, PTGS2, P2RY1, SEMA7A, SGK1 and SIX2, were highly up- or down-regulated. The expression of up-regulated genes was examined using the expression data of HNSCC patients obtained from The Cancer Genome Atlas (TCGA) database, and the expression of 5 genes, including PLAU, was found to be higher in cancer tissue than in solid normal tissue. An analysis of protein-protein interactions revealed that these 5 genes formed a dense network. A Cox regression analysis showed that high PLAU expression levels were associated with a poor prognosis in patients with TCGA-HNSCC. Furthermore, the prognostic impact correlated with tumour size and the presence or absence of lymph node metastasis. Collectively, these results suggest the potential of PLAU as a molecular prognostic marker in HNSCC patients. Further in vivo and in vitro studies are needed to verify the findings of this study.

Outline of Salivary Gland Pathogenesis of Sjögren's Syndrome and Current Therapeutic Approaches.

Sjögren's syndrome (SS) is an autoimmune disease characterized by the involvement of exocrine glands such as the salivary and lacrimal glands. The minor salivary glands, from which tissue samples may be obtained, are important for the diagnosis, evaluation of therapeutic efficacy, and genetic analyses of SS. In the onset of SS, autoantigens derived from the salivary glands are recognized by antigen-presenting dendritic cells, leading to the activation of T and B cells, cytokine production, autoantibody production by plasma cells, the formation of ectopic germinal centers, and the destruction of salivary gland epithelial cells. A recent therapeutic approach with immune checkpoint inhibitors for malignant tumors enhances the anti-tumor activity of cytotoxic effector T cells, but also induces SS-like autoimmune disease as an adverse event. In the treatment of xerostomia, muscarinic agonists and salivary gland duct cleansing procedure, as well as sialendoscopy, are expected to ameliorate symptoms. Clinical trials on biological therapy to attenuate the hyperresponsiveness of B cells in SS patients with systemic organ involvement have progressed. The efficacy of treatment with mesenchymal stem cells and chimeric antigen receptor T cells for SS has also been investigated. In this review, we will provide an overview of the pathogenesis of salivary gland lesions and recent trends in therapeutic approaches for SS.

Potential Role of the Intratumoral Microbiota in Prognosis of Head and Neck Cancer.

The tumor microbiome, a relatively new research field, affects tumor progression through several mechanisms. The Cancer Microbiome Atlas (TCMA) database was recently published. In the present study, we used TCMA and The Cancer Genome Atlas and examined microbiome profiling in head and neck squamous cell carcinoma (HNSCC), the role of the intratumoral microbiota in the prognosis of HNSCC patients, and differentially expressed genes in tumor cells in relation to specific bacterial infections. We investigated 18 microbes at the genus level that differed between solid normal tissue (n = 22) and primary tumors (n = 154). The tissue microbiome profiles of Actinomyces, Fusobacterium, and Rothia at the genus level differed between the solid normal tissue and primary tumors of HNSCC patients. When the prognosis of groups with rates over and under the median for each microbe at the genus level was examined, rates for Leptotrichia which were over the median correlated with significantly higher overall survival rates. We then extracted 35 differentially expressed genes between the over- and under-the-median-for-Leptotrichia groups based on the criteria of >1.5 fold and p < 0.05 in the Mann-Whitney U-test. A pathway analysis showed that these Leptotrichia-related genes were associated with the pathways of Alzheimer disease, neurodegeneration-multiple diseases, prion disease, MAPK signaling, and PI3K-Akt signaling, while protein-protein interaction analysis revealed that these genes formed a dense network. In conclusion, probiotics and specific antimicrobial therapy targeting Leptotrichia may have an impact on the prognosis of HNSCC.

Antimicrobial susceptibility surveillance of bacterial isolates recovered in Japan from odontogenic infections in 2013.

We report on the findings of the first antimicrobial susceptibility surveillance study in Japan of isolates recovered from odontogenic infections. Of the 38 facilities where patients representing the 4 groups of odontogenic infections were seen, 102 samples were collected from cases of periodontitis (group 1), 6 samples from pericoronitis (group 2), 84 samples from jaw inflammation (group 3) and 54 samples from phlegmon of the jaw bone area (group 4) for a total of 246 samples. The positivity rates of bacterial growth on culture were 85.3%, 100%, 84% and 88.9%, respectively, for groups 1, 2, 3 and 4. Streptococcus spp. isolation rates according to odontogenic infection group were 22% (group 1), 17.7% (group 3) and 20.7% (group 4). Anaerobic isolation rates were 66.9% (group 1), 71.8% (group 3) and 68.2% (group 4). Drug susceptibility tests were performed on 726 strains excluding 121 strains that were undergrown. The breakdown of the strains subjected to testing was 186 Streptococcus spp., 179 anaerobic gram-positive cocci, 246 Prevotella spp., 27 Porphyromonas spp., and 88 Fusobacterium spp. The isolates were tested against 30 antimicrobial agents. Sensitivities to penicillins and cephems were good except for Prevotella spp. The low sensitivities of Prevotella spp is due to ß-lactamase production. Prevotella strains resistant to macrolides, quinolones, and clindamycin were found. No strains resistant to carbapenems or penems were found among all strains tested. No anaerobic bacterial strain was resistant to metronidazole. Antimicrobial susceptibility testing performed on the S. anginosus group and anaerobic bacteria, which are the major pathogens associated with odontogenic infections, showed low MIC90 values to the penicillins which are the first-line antimicrobial agents for odontogenic infections; however, for Prevotella spp., penicillins combined with ß-lactamase inhibitor showed low MIC90 values.

Efficient Delivery and Replication of Oncolytic Virus for Successful Treatment of Head and Neck Cancer.

Head and neck cancer has been treated by a combination of surgery, radiation, and chemotherapy. In recent years, the development of immune checkpoint inhibitors (ICIs) has made immunotherapy a new treatment method. Oncolytic virus (OV) therapy selectively infects tumor cells with a low-pathogenic virus, lyses tumor cells by the cytopathic effects of the virus, and induces anti-tumor immunity to destroy tumors by the action of immune cells. In OV therapy for head and neck squamous cell carcinoma (HNSCC), viruses, such as herpes simplex virus type 1 (HSV-1), vaccinia virus, adenovirus, reovirus, measles virus, and vesicular stomatitis virus (VSV), are mainly used. As the combined use of mutant HSV-1 and ICI was successful for the treatment of melanoma, studies are underway to combine OV therapy with radiation, chemotherapy, and other types of immunotherapy. In such therapy, it is important for the virus to selectively replicate in tumor cells, and to express the viral gene and the introduced foreign gene in the tumor cells. In OV therapy for HNSCC, it may be useful to combine systemic and local treatments that improve the delivery and replication of the inoculated oncolytic virus in the tumor cells.

Transcription factors related to chondrogenesis in pleomorphic adenoma of the salivary gland: a mechanism of mesenchymal tissue formation.

In salivary gland pleomorphic adenoma, expression of extracellular matrix (ECM) substances indicates that tumor epithelial cells are becoming chondrogenic and will produce cartilage-like mesenchymal tissues. Sox9, the master transcription factor of chondrogenesis, is expressed in mouse salivary gland cells. To clarify the mechanism behind chondrogenesis in tumor epithelial cells, we examined the expression of transcription factors related to chondrogenesis in tumors and salivary glands. Reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and immunostaining were performed on pleomorphic adenoma tissues, salivary gland tissues, and human submandibular gland (HSG) cells. The mRNAs of essential transcription factors for chondrogenesis-Sox9, Sox6, and Sox5-were detected in both tumor and salivary gland tissues. The mRNAs of aggrecan and type II collagen-cartilage-specific ECM substances-were detected only in tumors. Sox9 and Sox6 proteins were colocalized in many epithelial cells in tumors and salivary glands. Tumor epithelial cells also possessed aggrecan protein and occasionally type II collagen protein. Moreover, mRNAs for transcription repressors of chondrogenesis δEF1 and AP-2α were detected in both tumors and salivary glands, whereas Twist1 mRNA was detected only in salivary glands and was at significantly low-to-undetectable levels in tumors. Twist1 protein was localized in the Sox9-expressing salivary gland cells. HSG cells expressed Sox9, Sox6, and Twist1, but not aggrecan or type II collagen, and thus were similar to salivary gland cells. Twist1 depletion by Twist1 siRNA led to the upregulation of aggrecan and type II collagen mRNA expression in HSG cells. In contrast, forced expression of Twist1, using Twist1 cDNA, resulted in the downregulation of both these genes. Taken together, these results indicate that salivary gland cells have a potential for chondrogenesis, and Twist1 depletion concomitant with neoplastic transformation, which would permit tumor epithelial cells to produce cartilage-like mesenchymal tissues in salivary gland pleomorphic adenoma.

Dermoid Cyst of the Lateral Neck Included Within the Submandibular Gland.

Dermoid cysts are benign lesions of congenital origin, and those in the head and neck region are usually present as a midline neck mass. They rarely appear in the lateral neck. This article describes the clinical presentation and histopathologic features of an extremely rare case of lateral dermoid cyst included within the submandibular gland in a 58-year-old man. The etiology of the cyst is also discussed.

Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability.

Hydroxyapatite (HA) or calcium carbonate (CaCO3) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO3-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO3-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO3-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO3-formed agarose at an early stage, and both HA- and CaCO3-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO3-formed agarose could potentially be employed for the repair of critical bone defects.

Surgery for pulmonary malignancies in patients with a previous history of head and neck squamous cell carcinoma.

PURPOSE: To examine the perioperative and long-term outcomes of surgery for malignancies of the lungs in patients with a history of head and neck squamous cell carcinoma (HNSCC) and to evaluate the risk factors associated with postoperative complications. METHODS: The data of 39 patients with a history of HNSCC who underwent pulmonary resection were reviewed. The perioperative and long-term outcomes were analyzed. RESULTS: Eight patients (21%) had difficult airways, and nine patients (23%) developed postoperative complications. A low body mass index (<18.5), a history of malignancy besides HNSCC and chronic obstructive pulmonary disease were each found to be significantly associated with the development of postoperative complications. The 5-year survival rate of all patients was 80%. CONCLUSIONS: The airway management of patients with a history of HNSCC should be carefully undertaken. Preoperative assessment of their nutritional status and careful prevention of air leakage during surgery are important. Because favorable outcomes can be achieved, aggressive surgical management should be considered for the treatment of pulmonary malignancies in patients with a history of HNSCC.

Involvement of hydrogen peroxide in safingol-induced endonuclease G-mediated apoptosis of squamous cell carcinoma cells.

Safingol, a L-threo-dihydrosphingosine, induced the nuclear translocation of a mitochondrial apoptogenic mediator--endonuclease G (endo G)--and apoptosis of human oral squamous cell carcinoma (SCC) cells. Upstream mediators remain largely unknown. The levels of hydrogen peroxide (H2O2) in cultured oral SCC cells were measured. Treatment with safingol increased intracellular H2O2 levels but not extracellular H2O2 levels, indicating the production of H2O2. The cell killing effect of safingol and H2O2 was diminished in the presence of reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC). Dual staining of cells with annexin V and propidium iodide (PI) revealed that apoptotic cell death occurred by treatment with H2O2 and safingol. The number of apoptotic cells was reduced in the presence of NAC. In untreated cells, endo G distributed in the cytoplasm and an association of endo G with mitochondria was observed. After treatment with H2O2 and safingol, endo G was distributed to the nucleus and cytoplasm, indicating the nuclear translocation of the mitochondrial factor. NAC prevented the increase of apoptotic cells and the translocation of endo G. Knock down of endo G diminished the cell killing effect of H2O2 and safingol. These results suggest that H2O2 is involved in the endo G-mediated apoptosis of oral SCC cells by safingol.

Sequence of a fusogenic herpes simplex virus, RH2, for oncolytic virotherapy.

RH2 is a novel oncolytic herpes simplex virus type 1 (HSV-1) produced by simultaneous infection with neurovirulent γ134.5 gene-deficient HSV-1 R849 derived from strain F and the spontaneously occurring, fusogenic HSV-1 HF in cell culture. The genome of RH2 was studied using Genome Sequencer FLX. RH2 comprised 149 64 bp and it was shown that the lacZ gene was inserted into the γ134.5 gene of R849. Comparison of ORFs revealed that RH2 had 100 % identity with strain F in 21/58 unique long (UL) genes (36.2%) and 1/13 unique short (US) genes (7.7%). RH2 had 100% amino acid identity with HF10 in 24/58 UL genes (41.4%) and 9/13 US genes (69.2%). Twelve genes, including UL27 (gB), US4 (gG) and UL6 (gD), had amino acid changes unique to RH2. Amino acid changes in gB occurred at positions 459 (T→A) and 817 (L→P). Other unique features were the amino acids missing in UL36 (VP1/2) and UL46 (VP11/12). Thus, RH2 is an HF10-based vector preserving the fusogenic amino acid changes of gB but lacking the γ134.5 gene. RH2 is expected to be a version of HF10 useful for the treatment of brain tumours as well as oral squamous cell carcinoma. Spontaneously occurring HSV-1 mutants may also be useful clinically, as their genome sequences can easily be determined by this genome sequencing system.

Oral Immune-Related Adverse Events Caused by Immune Checkpoint Inhibitors: Salivary Gland Dysfunction and Mucosal Diseases.

Conventional chemotherapy and targeted therapies have limited efficacy against advanced head and neck squamous cell carcinoma (HNSCC). The immune checkpoint inhibitors (ICIs) such as antibodies against CTLA-4, PD-1, and PD-L1 interrupt the co-inhibitory pathway of T cells and enhance the ability of CD8+ T cells to destroy tumors. Even in advanced HNSCC patients with recurrent diseases and distant metastasis, ICI therapy shows efficiency and become an effective alternative to conventional chemotherapy. However, as this therapy releases the immune tolerance state, cytotoxic CD8+ T cells can also attack organs and tissues expressing self-antigens that cross-react with tumor antigens and induce immune-related adverse events (irAEs). When patients with HNSCC are treated with ICIs, autoimmune diseases occur in multiple organs including the skin, digestive tract, endocrine system, liver, and respiratory tract. Treatment of various malignancies, including HNSCC, with ICIs may result in the appearance of oral irAEs. In the oral cavity, an oral lichenoid reaction (OLR) and pemphigoid develop. Sicca syndrome also occurs in association with ICIs, affecting the salivary glands to induce xerostomia. It is necessary to elucidate the pathogenic mechanisms of these intractable diseases that are not seen with conventional therapy. Early diagnosis and appropriate approaches to irAEs are needed for efficient treatment of advanced HNSCC by ICIs.

RPAP3 enhances cytotoxicity of doxorubicin by impairing NF-kappa B pathway.

Activation of anti-apoptotic gene transcription by NF-κB (nuclear factor-kappa B) has been reported to be linked with a resistance of cancer cells against chemotherapy. NEMO (NF-κB essential modulator) interacts with a number of proteins and modulates the activity of NF-κB pathway. In this study, we revealed that RPAP3 (RNA polymerase II-associated protein 3) possesses an activity to bind with NEMO and to inhibit the ubiquitination of NEMO and that RPAP3 enhances doxorubicin-induced cell death in breast cancer cell line T-47D through the marked impairment of NF-κB pathway. These results indicate that RPAP3 may be a novel modulator of NF-κB pathway in apoptosis induced by anti-cancer chemotherapeutic agents.

Lymphatic vessels and related factors in adenoid cystic carcinoma of the salivary gland.

Adenoid cystic carcinoma of the salivary gland preferentially metastasizes to distant organs. It rarely metastasizes to lymph nodes. Recently, lymphangiogenesis has been associated with lymph node metastasis. Therefore, lymphangiogenesis in adenoid cystic carcinoma was evaluated from the number of lymphatic vessels and the expression of lymphangiogenic factors. Immunohistochemistry and molecular analysis were performed on clinical materials (29 cases for immunohistochemistry and 9 cases for molecular analysis). Normal submandibular gland was used as a negative control of lymphangiogenesis (10 cases for immunohistochemistry and 5 cases for molecular analysis). In adenoid cystic carcinoma, podoplanin-positive lymphatic vessels were small and often constricted, and localized to the tumor periphery. They did not have Ki67-positive endothelial cells. The lymphatic vessel density of the tumor did not exceed that of the salivary gland. By reverse transcriptase-polymerase chain reaction, adenoid cystic carcinoma and the salivary gland expressed vascular endothelial growth factor receptor-3 (VEGFR-3) similarly but VEGF-C and VEGF-D differently. Adenoid cystic carcinoma expressed VEGF-C, whereas the salivary gland expressed both VEGF-C and VEGF-D. VEGF-C was weak in adenoid cystic carcinoma and strong in the salivary gland. Real-time reverse transcriptase-polymerase chain reaction of VEGF-C showed that the ratio of the tumor to the salivary gland was 1 to 30 (P<0.01). Immunohistochemistry barely detected VEGF-C in adenoid cystic carcinoma. VEGF-C was expressed faintly by the tumor cells. VEGF-C and VEGF-D were detected in the serous acinar and duct cells and in the duct contents in the salivary gland. VEGFR-3 appeared to be expressed by lymphatic vessels in both adenoid cystic carcinoma and the salivary gland. These results indicate that lymphangiogenesis does not occur in adenoid cystic carcinoma. This condition would lead to the uncommon lymphatic metastasis.

A novel fusogenic herpes simplex virus for oncolytic virotherapy of squamous cell carcinoma.

BACKGROUND: R849 is a neurovirulent γ134.5 gene-deficient form of herpes simplex virus type 1 (HSV-1) and has LacZ genes at the deleted sites of the γ134.5 gene. HF is a spontaneously occurring, fusogenic HSV-1 strain. The purpose of this work was to generate a virus that has the syncytial character of HF, while preserving the γ134.5 gene inactivation profile of R849 virus. RESULTS: Vero cells were infected with R849 and HF simultaneously and two viruses, RH1 and RH2, expressing the LacZ gene and inducing extensive cell fusion were selected. A polymerase chain reaction (PCR)-based analysis suggested that one copy of the γ134.5 gene is lost in RH1, whereas both copies are lost in RH2, and that the γ134.5 gene is replaced by a R849-derived DNA fragment with the LacZ gene. These viruses produced larger plaques and more progeny than the parental viruses. Infection with RH2 decreased the viability of oral squamous cell carcinoma (SCC) cells most strongly. When RH2 was injected into xenografts of oral SCC in nude mice, multinucleated cells were produced and the growth of the tumors was suppressed significantly. CONCLUSION: These results indicate that novel oncolytic HSV-1 vectors can be produced with the genetic background of the oncolytic HSV-1 HF, and that RH2 is deficient in γ134.5 genes and shows extensive cytopathic effects in oral SCC cells. RH2 may be useful in oncolytic virotherapy for oral SCC.

Effect of ultrasound on herpes simplex virus infection in cell culture.

BACKGROUND: Ultrasound has been shown to increase the efficiency of gene expression from retroviruses, adenoviruses and adeno-associated viruses. The effect of ultrasound to stimulate cell membrane permeabilization on infection with an oncolytic herpes simplex virus type 1 (HSV-1) was examined. RESULTS: Vero monkey kidney cells were infected with HSV-1 and exposed to 1 MHz ultrasound after an adsorption period. The number of plaques was significantly greater than that of the untreated control. A combination of ultrasound and microbubbles further increased the plaque number. Similar results were obtained using a different type of HSV-1 and oral squamous cell carcinoma (SCC) cells. The appropriate intensity, duty cycle and time of ultrasound to increase the plaque number were 0.5 W/cm², 20% duty cycle and 10 sec, respectively. Ultrasound with microbubbles at an intensity of 2.0 W/cm², at 50% duty cycle, or for 40 sec reduced cell viability. CONCLUSION: These results indicate that ultrasound promotes the entry of oncolytic HSV-1 into cells. It may be useful to enhance the efficiency of HSV-1 infection in oncolytic virotherapy.

Prognostic association of starvation-induced gene expression in head and neck cancer.

Autophagy-related genes (ARGs) have been implicated in the initiation and progression of malignant tumor promotion. To investigate the dynamics of expression of genes, including ARGs, head and neck squamous cell carcinoma (HNSCC) cells were placed under serum-free conditions to induce growth retardation and autophagy, and these starved cells were subjected to transcriptome analysis. Among the 21 starvation-induced genes (SIGs) located in the autophagy, cell proliferation, and survival signaling pathways, we identified SIGs that showed prominent up-regulation or down-regulation in vitro. These included AGR2, BST2, CALR, CD22, DDIT3, FOXA2, HSPA5, PIWIL4, PYCR1, SGK3, and TRIB3. The Cancer Genome Atlas (TCGA) database of HNSCC patients was used to examine the expression of up-regulated genes, and CALR, HSPA5, and TRIB3 were found to be highly expressed relative to solid normal tissue in cancer and the survival rate was reduced in patients with high expression. Protein-protein interaction analysis demonstrated the formation of a dense network of these genes. Cox regression analysis revealed that high expression of CALR, HSPA5, and TRIB3 was associated with poor prognosis in patients with TCGA-HNSCC. Therefore, these SIGs up-regulated under serum starvation may be molecular prognostic markers in HNSCC patients.

Inhibitors of Ceramide- and Sphingosine-Metabolizing Enzymes as Sensitizers in Radiotherapy and Chemotherapy for Head and Neck Squamous Cell Carcinoma.

In the treatment of advanced head and neck squamous cell carcinoma (HNSCC), including oral SCC, radiotherapy is a commonly performed therapeutic modality. The combined use of radiotherapy with chemotherapy improves therapeutic effects, but it also increases adverse events. Ceramide, a central molecule in sphingolipid metabolism and signaling pathways, mediates antiproliferative responses, and its level increases in response to radiotherapy and chemotherapy. However, when ceramide is metabolized, prosurvival factors, such as sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and glucosylceramide, are produced, reducing the antitumor effects of ceramide. The activities of ceramide- and sphingosine-metabolizing enzymes are also associated with radio- and chemo-resistance. Ceramide analogs and low molecular-weight compounds targeting these enzymes exert anticancer effects. Synthetic ceramides and a therapeutic approach using ultrasound have also been developed. Inhibitors of ceramide- and sphingosine-metabolizing enzymes and synthetic ceramides can function as sensitizers of radiotherapy and chemotherapy for HNSCC.

Induction of apoptosis of detached oral squamous cell carcinoma cells by safingol. Possible role of Bim, focal adhesion kinase and endonuclease G.

The protein kinase C (PKC) inhibitor safingol increased rounding and detachment of human oral squamous cell carcinoma (SCC) cells in monolayer cultures. When dissociated cells were incubated in the presence of safingol, cell adhesion was prevented and cell viability was lost gradually, while most cells survived in the absence of safingol even if their attachment was blocked by coating the culture plates with polyhydroxyethyl methacrylate. Flow cytometric analysis and agarose gel electrophoresis of cellular DNA revealed an increase in the proportion of sub-G(1) cells and DNA fragmentation, indicating that safingol induced apoptosis of dissociated cells. During the induction of apoptosis in cell suspensions by safingol, there was an increase of the pro-apoptotic BH-3 only protein Bim and decrease of pro-survival Bcl-2 family proteins Bcl-xL and mitochondrial pro-apoptogenic factor endonuclease G translocated to the nucleus. The level of phosphorylated focal adhesion kinase (FAK) required for cell survival also rapidly decreased, followed by a decrease in the protein level. The introduction of siRNA against PKCalpha into SAS cells resulted in an increase of Bim, a decrease of Bcl-xL, the translocation of endonuclease G, and a decrease in the phosphorylation of FAK. These results suggest that Bim, Bcl-xL, FAK and endonuclease G are involved in safingol-induced apoptosis of detached oral SCC cells. Safingol can be used to induce apoptosis with cell detachment, anoikis, of oral SCC cells.

Label-retaining cells in the rat submandibular gland.

To identify stem cells in salivary glands, label-retaining cells (LRCs) were established in rat submandibular glands. Developing and regenerating glands were labeled with bromodeoxyuridine (BrdU). To cause gland regeneration, the glands were injured by duct obstruction. BrdU LRCs were observed in all the parenchymal structures except for the acinus of the glands labeled during regeneration. Among these LRCs, a few, but not many, expressed neither keratin18 (K18; an acinar/duct cell marker) nor alpha-smooth muscle actin (alphaSMA; a myoepithelial cell marker), and thus were putative stem cells. These (K18 and alphaSMA)(neg) LRCs were invariably observed in the intercalated duct and the excretory duct. In the intercalated duct, they were at the proximal end bordering the acinus (the neck of the intercalated duct). Next, to test the above identification, gland extirpation experiments were performed. LRCs were established by labeling developing glands with iododeoxyuridine (IdU) in place of BrdU. Removal of one submandibular gland forced the IdU-LRCs in the remaining gland to divide. They were labeled with chlorodeoxyuridine (CldU). The (K18 and alphaSMA)(neg) LRCs in the neck of the intercalated duct and in the excretory duct did not change in number or in IdU label. The CldU label appeared in these cells and then disappeared. These results indicate that the (K18 and alphaSMA)(neg) LRCs have divided asymmetrically and are thus considered salivary gland stem cells.