Yasutaka Hayashido

Constitutive activating mutation of the FGFR3b in oral squamous cell carcinomas

A G to T mutation at nucleotide position 2128 in the human FGFR3b coding region resulting in a Cys for Gly substitution (G697C) in the tyrosine kinase domain was observed in 62% (44/71) of oral squamous cell carcinomas (OSCC) examined. Immunostained FGFR3b was found in the cytoplasm of prickle cells in normal epithelia, and FGFR3b was localized in the cytoplasm and nucleus in non‐FGFR3b mutant OSCC. Overexpressed FGFR3b protein on plasma membranes was noted in OSCC bearing the FGFR3b mutation. Enhanced tyrosine kinase activity of G697CFGFR3b was confirmed. Our results indicate that G697C is an activating mutation causing constitutive ligand‐independent FGFR3b signaling. This mutation may be involved in the progression of OSCC and thus the FGFR3b coding sequence may have diagnostic or prognostic value for OSCC.

Isolation and serum-free culture of epithelial cells derived from epithelial rests of Malassez in human periodontal ligament

SummaryTo study biological character and function of epithelial rests of Malassez (ERM) in human periodontal ligament, we have developed a serum-free culture system of epithelial cells (ME) derived from ERM. The mitogenic effects of fibroblast growth factor (FGF)-1, FGF-2, and FGF-7/keratinocyte growth factor (KGF) on ME, human periodontal ligament-derived fibroblasts (PLF), human oral epithelial cells (OE), and human submandibular gland-derived epithelial cells (SGE) were investigated under a serum-free culture condition. FGF-1 and FGF-7/KGF stimulated the growth of both ME and SGE but FGF-2 had no effect. On the other hand, FGF-1, FGF-2, and FGF-7/KGF increased the OE proliferation. These results suggested that the divergent requirement of FGF ligands among these cells would be attributed to the different expression pattern of FGF receptor (FGFR) messenger ribonucleic acid (mRNA) isotypes. Therefore, we examined the expression of FGFR isotypes in these cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of ME- and SGE-derived mRNAs revealed the presence of mRNA encoding FGFR2-IIIb, a high affinity receptor for FGF-1 and FGF-7/KGF. However, no mRNAs for other FGFR isotypes were detected in both ME and SGE. On the contrary, OE expressed FGFR1-IIIc, FGFR3-IIIb, and FGFR4 mRNAs as well as FGFR2-IIIb. These results indicate that FGF binding sites on ME dominantly bind to FGF-1 and FGF-7/KGF, which transduce their signals via FGFR2-IIIb. Immunohistochemical analysis, PCR-Southern, ribonuclease protection assay (RPA), and Western blotting revealed that PLF expressed FGF-7/KGF mRNA and its peptide. These observations suggest that FGF-7/KGF might mediate epithelial-mesenchymal interactions between ME and PLF to maintain normal structure and function of periodontal ligament.

Mutations in the human homologue of the Drosophila segment polarity gene patched in oral squamous cell carcinoma cell lines

SummaryIn the present study, we have analyzed tumor deoxyribonucleic acid from oral squamous cell carcinoma (OSCC) cells for patched mutations using an exon-by-exon single strand conformation polymorphism assay and direct sequencing. We found two missense mutations which affected the conserved residue in the transmembrane domains of the gene product and in the intracellular loop at the C-terminal residue implicated in regulating the smoothened molecule. In addition, we demonstrated that the N-terminal fragment of sonic hedgehog (Shh-N) stimulates the growth of normal epithelial cells, the OSCC cell line, NA, and the salivary gland adenocarcinoma cell lines, HSG and HSY, which have no detectable mutation in patched. On the other hand, Shh has no effect on human SCC cells (UE, KA, KO, NI, A431 cells) that have mutations in patched. These results strongly suggest that an Shh-patched signaling is involved in the cell growth of oral epithelial cells and in the tumorigenesis of OSCCs.

EXPRESSION OF FIBROBLAST GROWTH FACTOR RECEPTOR GENES IN HUMAN HEPATOMA-DERIVED CELL LINES

SummaryThe fibroblast growth factor (FGF) function has been considered to contribute to various human tumors and malignant growth of neoplasm. Hepatocellular carcinoma (HCC) is a typical hypervascular tumor, and it is suggested that FGF may be involved in hepatocarcinogenesis. Therefore, the relationship between the progression of HCC and expression of FGFs and FGF receptors (FGFRs) was evaluated in this study. We investigated the expression of messenger ribonucleic acids (mRNAs) of FGFs and FGFRs by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in eight human hepatoma-derived cell lines (Hep3B, HLE, HLF, HUH6, HUH7, KIM1, Li7, and PLC/PRF/5), one hepatoblastome-derived cell line (HepG2), and human primary hepatocytes. In addition, effects of FGF-1, FGF-2, and FGF-7 on the growth of hepatoma-derived cell lines were studied in serum-free defined culture conditions. An RT-PCR analysis revealed that all cell lines except PLC/PRF/5 expressed all FGFR MRNAs: FGF-R1 (IIIc),-R2 (IIIb),-R3 (IIIb),-R3 (IIIc), and-R4 mRNAs. In contrast, human primary hepatocytes expressed FGF-R1 (IIIc),-R3 (IIIc), and-R4 mRNAs but not mRNAs of FGF-R2 (IIIb),-R2 (IIIc), and-R3 (IIIb). All cell lines except HUH6 and HUH7 expressed FGF-1 and FGF-2 mRNAs. Addition of exogenous FGF-1 or FGF-2 (or both) to culture stimulated cell proliferation in several cell lines, but FGF-7 exhibited no growth stimulation in all cells. Hepatoma cells may posses a proliferation mechanism regulated by an autocrine mechanism, a paracrine mechanism, or both, which are mediated by FGF-1/FGFR or FGF-2/FGFR (or both). In addition, a gain of FGF-R2 (IIIb),-R2 (IIIc), and-R3 (IIIb) may be associated with malignant transformation of liver tumor and may eventually serve as useful diagnostic and prognostic indicators.

Overexpression of integrin αv facilitates proliferation and invasion of oral squamous cell carcinoma cells via mek/erk signaling pathway that is activated by interaction of integrin αvβ8 with type I collagen

To examine the role of integrin αv subunit in the progression of squamous cell carcinoma (SCC), oral SCC cells were stably transfected with integrin αv cDNA. Integrin αv transfectants exhibited the enhancement of proliferation on type Ⅰ collagen, and seemed to have a high ability to invade type Ⅰ collagen gel. Overexpression of integrin αv led to rapid phosphorylation of focal adhesion kinase (FAK), mitogen‑activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal‑regulated kinase 1/2 (ERK1/2) in SCC cells on type Ⅰ collagen. The downregulation of integrin β8 in integrin αv transfectants by its specific antisense oligonucleotide led to a decrease in type Ⅰ collagen‑stimulated activation of FAK and the MEK/ERK signaling pathway, and also suppressed the proliferation on type Ⅰ collagen and the invasiveness into type Ⅰ collagen gel. Moreover, the expression of integrin β8 was induced following transfection with integrin αv cDNA. These results indicated that the overexpression of integrin αv induces integrin αvβ8 heterodimer formation and the binding of integrin αvβ8 to type Ⅰ collagen might enhance the proliferation and invasion of SCC cells via the activation of the MEK/ERK signaling pathway.

Ectomesenchymal chondromyxoid tumor of the tongue: insights on histogenesis

OBJECTIVE The objective of this study was to investigate the histogenesis of ectomesenchymal chondromyxoid tumors (ECTs) of the tongue. STUDY DESIGN The biochemical characteristics of a rarely occurring tumor of the tongue were analyzed by immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR), and its biological properties were assessed in primary culture in serum-free media. RESULTS Immunohistochemistry showed that the tumor cells were strongly positive for vimentin, S-100, and glial fibrillary acidic protein (GFAP), but negative for cytokeratin and epithelial membrane antigen. In primary cultures, the cells derived from the ECT were morphologically similar to neuronal cells and expressed Nanog, GFAP, and MAP2. RT-PCR analysis of the surgical specimen was positive for OCT3/4, Sox2, Nanog, MAP2, and CD105 mRNAs. CONCLUSIONS The results of the present study indicate that ECTs originate from the ectomesenchymal cells of the neural crest and are similar in their molecular and biological characteristics to undifferentiated mesenchymal stem cells.

Eldecalcitol (ED-71), an analog of 1α,25-dihydroxyvitamin D3 as a potential anti-cancer agent for oral squamous cell carcinomas

We have previously reported that 1,25(OH)2D3 inhibits NF-κB activity and thus inhibits growth of OSCC cells in serum-free culture and down-regulates HBp17/FGFBP-1 expression, which is important for cancer cell growth and angiogenesis. Here, we have investigated the effects of ED-71, an analog of vitamin D3 (VD) on OSCC cell lines in serum-free culture. It is known that ED-71 has a stronger inhibitory effect on bone resorption compared to VD and other VD analogs. To the best of our knowledge, there was no report examining the potential of ED-71 as an anti-cancer agent for OSCC. We found that ED-71 is able to inhibit the growth of cancer cell lines at a concentration of hundred times lower than calcitriol. As Cyp24A1 was reportedly induced in cancer cells, we measured the expression of CYP24A1 in OSCC cell lines (NA and UE), A431 epidermoid carcinoma and normal fibroblast cell (gfi) in serum-free culture. As a result, CYP24A1 mRNA and the protein expression in the OSCC cells treated with ED-71 increased in a dose-dependent manner. However, in vivo experiment, in which the A431 cells were implanted in mice, tumor formation was reduced by the ED-71 treatment with no significant difference between Cyp24A1 expression in the tumors of ED-71-treated and control group, as analyzed by western blotting and immunohistochemistry. These results suggest that ED-71 is a potential anti-cancer agent for OSCC.

Developmental Signaling Disorders in Craniofacial Anomalies and Cancers

Normal human development requires the precise functioning and coordination of many complex pathways. Abnormalities in these signaling cascades often result in developmental perturbations, giving rise to congenital anomalies and cancers. There are 21,787 genes in each human nucleus, different gene subsets are expressed in different cell types, and different gene networks make different signal cascades. Among a large number of genes, in this review, we describe signaling disorders of sonic hedgehog and its receptor, patched-1 ; Tie2 ; fi broblast growth factor receptor in craniofacial anomalies and oral cancers.

Eldecalcitol (ED-71), an analog of 1α,25(OH) 2 D 3 , inhibits the growth of squamous cell carcinoma (SCC) cells in vitro and in vivo by down-regulating expression of heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) and FGF-2

Heparin-binding protein 17 (HBp17)/fibroblast growth factor-binding protein-1 (FGFBP-1) was first purified from medium conditioned by A431 cells for its capacity to bind to fibroblast growth factors 1 and 2 (FGF-1 and -2). Among FGF family members, FGF-2 is a potent mitogen for various cell types, including vascular endothelial cells, fibroblasts, and cancer cells such as oral squamous cell carcinoma (OSCC) cells. Besides being well known in bone metabolism, the active form of vitamin D3, i.e., 1α,25(OH)2D3 (1,25D3), was reported to have protective effects for heart disease and cancer. Previously, we reported that 1,25D3 inhibited HBp17/FGFBP-1 expression in OSCC cell lines through NF-κB inhibition (IκBα activation) and resulted in the inactivation of FGF-2. In this study, we examined the potential anti-tumor effect of ED-71, an analog of 1α,25(OH)2D3, for squamous cell carcinoma cells in vitro and in vivo. The cell lines used were OSCC cell lines (NA—HO-1-n-1 and UE—HO-1-u-1), established from oral cancer patients in our laboratory, and an epidermoid carcinoma/SCC cell line (A431). The growth assay in serum-free culture revealed that ED-71 inhibited the growth of the cancer cell lines in a dose-dependent manner. In addition, ED-71 suppressed HBp17/FGFBP-1 expression by inhibiting the NF-κB pathway as did 1,25D3. Furthermore, a luciferase reporter assay revealed that the promoter activity of HBp17/FGFBP-1 (region between −217 and +61) was down-regulated by ED-71. Oral administration of ED-71 significantly inhibited the growth of A431-derived tumors in athymic nude mice. Immunohistochemical analysis revealed that the expression of HBp17/FGFBP-1, FGF-2, CD31, and Ki-67 in the tumors of ED71-treated group was down-regulated in comparison to control. These results suggest that ED-71 possesses potential anti-tumor activity for SCCs both in vitro and in vivo. This compound may act directly on the tumor cells or on endothelial cells by modulating the tumor microenvironment.

Weekly paclitaxel plus cetuximab reduces the lung metastasis of adenoid cystic carcinoma arising from the salivary gland

Abstract Adenoid cystic carcinoma (ACC) of the salivary gland often metastasizes to the lungs. It has previously been reported that the combination chemotherapy of weekly paclitaxel plus cetuximab is effective for the treatment of lung metastasis from ACC of the salivary gland. Here, we report a case in which a regimen of this combination therapy provided an antitumor treatment for lung metastatic ACC arising from the minor salivary gland of the hard palate.