Evaluation of immunohistochemical expression of TWIST in oral epithelial dysplasia and squamous cell carcinoma.

INTRODUCTION: The major transcription factor, which modulates the epithelial-mesenchymal transition in different types of cancers, is known as TWIST oncogene. It binds to the promoter of E-cadherin and suppresses its transcription. The current study aims to assess the expression of TWIST protein in oral squamous cell carcinoma (OSCC), epithelial dysplasia (ED), and normal oral mucosa to verify whether such protein is useful as a marker in oral epithelium malignant transformation. METHODS: Thirty-five paraffin-embedded tissue samples of oral lesions with ED and OSCC and five samples of normal oral mucosa were immuostained with anti-TWIST antibody using the streptavidin peroxidase method. RESULTS: TWIST expression was negative in all cases of normal oral mucosa, whereas all cases of ED and OSCC showed positive immunoreactivity to TWIST varied from weak to strong expression. In ED, there was a significant difference between severe dysplasia and the other two types (P = 0.03). TWIST expression had no significant relationship with the clinical parameters of OSSC clinical stage and grade (degree of differentiation). Only two cases of OSCC with lymph node metastasis showed strong nuclear TWIST expression. Intergroups assessment indicated a significant increase of TWIST expression in OSCC compared to ED (P = 0.000). CONCLUSION: A significant increase of TWIST expression in OSCC compared to ED may suggest its role in carcinogenesis, it may be a useful marker in malignant transformation of oral epithelium. Therefore, TWIST might be an important target for therapeutic approaches in patients with OSCC, which requires further investigations.

Parenchymal-stromal switching for extracellular matrix production on invasion of oral squamous cell carcinoma.

It is poorly understood which cell type, tumor cells, or stromal cells are responsible for the production of extracellular matrix molecules in the neoplastic stroma. We studied the expression of 4 extracellular matrix molecules at the protein and messenger RNA levels in monocellular and 2 kinds of coculture systems between human squamous cell carcinoma (ZK-1) and fibroblast (OF-1) cell lines, which may correspond to carcinoma in situ and squamous cell carcinoma, respectively. Squamous cell carcinoma and carcinoma in situ tissue sections were also investigated by immunohistochemistry and in situ hybridization for extracellular matrix. Immunohistochemically, perlecan and tenascin C were localized in carcinoma cells in carcinoma in situ, whereas they were in the stromal space in squamous cell carcinoma. In monocellular culture conditions, expression levels for perlecan, tenascin C, and laminin were more predominant in ZK-1 than in OF-1, although those for fibronectin were more enhanced in OF-1. However, these extracellular matrix expression levels of OF-1 were elevated, whereas those of ZK-1 dropped when they were in coculture conditions. The differences between ZK-1 and OF-1 were significantly more evident in direct contact (ZK-1/OF-1, 56%-22%) than in indirect contact (63%-39%). These results indicate that oral squamous cell carcinoma cells produce extracellular matrix in the absence of stromal fibroblasts (or in carcinoma in situ) and that they stop producing extracellular matrix in the presence of fibroblasts (or in squamous cell carcinoma). It is hence suggested that stromal fibroblasts after direct contact with invading squamous cell carcinoma cells are more responsible than squamous cell carcinoma cells for the formation of neoplastic stroma, whereas carcinoma in situ cells have to produce and deposit extracellular matrix by themselves to form intraepithelial microstromal spaces.

Perlecan, a basement membrane-type heparan sulfate proteoglycan, in the enamel organ: its intraepithelial localization in the stellate reticulum.

The localization and biosynthesis of perlecan, a basement membrane-type heparan sulfate proteoglycan, were studied in developing tooth germs by using murine molars in neonatal and postnatal stages and primary cultured cells of the enamel organ and dental papilla to demonstrate the role of perlecan in normal odontogenesis. Perlecan was immunolocalized mainly in the intercellular spaces of the enamel organ as well as in the dental papilla/pulp or in the dental follicle. By in situ hybridization, mRNA signals for perlecan core protein were intensely demonstrated in the cytoplasm of stellate reticulum cells and in dental papilla/pulp cells, including odontoblasts and fibroblastic cells in the dental follicle. Furthermore, the in vitro biosyntheses of perlecan core protein by the enamel organ and dental papilla/pulp cells were confirmed by immunofluorescence, immunoprecipitation, and reverse transcriptase-polymerase chain reaction. The results indicate that perlecan is synthesized by the dental epithelial cells and is accumulated in their intercellular spaces to form the characteristic stellate reticulum, whose function is still unknown.

Establishment and characterization of new cell lines derived from melanotic neuroectodermal tumor of infancy arising in the mandible.

Three cell systems (MINT1/2/3) derived from a melanotic neuroectodermal tumor of infancy (MNTI) arising in the mandible of a 1-month-old newborn boy have been established, and their cytological natures have been characterized. The cells had immunopositivities for pan-keratin, vimentin, neuron-specific enolase, S-100 protein and melanoma-associated antigen (HMB-45). These immunohistochemical phenotypes were basically the same as those observed in tissue sections, in which, synaptophysin, myelin basic protein, c-myc gene products, carcinoembryonic antigen, and epithelial membrane antigen were also immunolocalized in tumor cells. Karyotyping analyzes revealed that the chromosome numbers of the three cell systems ranged from 60 to 67 with 3n ploidies, and that there were many structural aberrations, such as del(11)(q13), del(22)(q13), add(2)(p11), add(7)(q22), extra copies for chromosomes 1, 2, 3, 5, 7, 9, 10, 11, 12, 16, 20, and 22, der(9)t(9;13)(p13;q12)add(9)(q34), and der(13;21)(q10;q10), which were shared by the three cell systems, while der(19)t(11;19)(q13;p13) was found in MINT1 and MINT3. When stimulated by endothelin-3 and vitamin D(3), the cells had spinous cell shapes with immunopositivities for HMB-45, neurofilament protein and glial fibrillary acidic protein, which indicated more neural differentiation. The established cell systems will be useful for further investigation on the molecular and genetic basis of MNTI to understand its pathogenesis, which is largely unknown.

Vascular endothelial cell participation in formation of lymphoepithelial lesions (epi-myoepithelial islands) in lymphoepithelial sialadenitis (benign lymphoepithelial lesion).

Lymphoepithelial lesions (LELs, or epi-myoepithelial islands) in lymphoepithelial sialadenitis (LESA, or benign lymphoepithelial lesion) of the salivary gland are known to be mainly composed of duct epithelial cells. However, other constituent cells are poorly characterized. Formalin-fixed paraffin sections obtained from six surgical specimens of LESA were examined using immunohistochemistry for cytoskeletal proteins, inflammatory cells, vascular endothelial cells, and extracellular matrix (ECM) molecules as well as by in situ hybridization for ECM molecules. In addition to keratin-immunopositive (+) duct-like epithelial cells, there were CD31/CD34+ vascular endothelial cells-which were either scattered in a singular fashion, in formed sheets, or in tubular structures-, CD20+ B lymphocytes, CD45RO+ T lymphocytes, and CD68 macrophages in the LELs. ECM molecules, such as heparan sulfate proteoglycan and tenascin, were immunolocalized in hyaline materials in the LELs. Their mRNAs were demonstrated mainly in endothelial cells and, to a lesser extent, in lympho-monocytic cells around hyaline materials, but were not as evident in epithelial constituent cells of LELs. The results indicate that endothelial cells as well as inflammatory cells are important constituents of the LELs, and the hyaline ECM cores mainly result from the intra-LEL angiogenesis by endothelial cells with the assistance of inflammatory cells. This intra-LEL vasculature seems to support regeneration and proliferation of salivary epithelial remnant cells.