Local invasiveness of ameloblastoma. Role played by matrix metalloproteinases and proliferative activity.

AIMS: Ameloblastoma is an odontogenic neoplasm characterized by local invasiveness and recurrence. In this study we analysed the role played by matrix metalloproteinases (MMPs) in the local invasiveness of ameloblastoma. We also attempted to establish a relationship between the presence of MMPs and the proliferative activity of ameloblastoma cells. METHODS AND RESULTS: Immunohistochemistry was carried out to detect different MMPs in formalin-fixed paraffin-embedded samples of human ameloblastoma. Immunohistochemistry, however, does not establish whether a given MMP is latent or active. To address this point, we carried out biochemical methods, namely zymography and Western blotting. Our results showed expression of latent and active forms of MMPs 1, 2 and 9 in ameloblastoma. These enzymes may digest bone matrix and release mitogenic factors, which would increase tumour proliferation. This possibility prompted us to study the proliferation of ameloblastoma cells located in close proximity to bone. Silver-stained nucleolar organizer region morphometry revealed that ameloblastoma cells in the vicinity of bone show increased proliferation, when compared with controls. CONCLUSIONS: Our results suggest an interdependent mechanism involving MMPs and proliferation of ameloblastoma cells, which may contribute to the local invasiveness of this tumour.

Brefeldin-A induces apoptosis in human adenoid cystic carcinoma cultured cells.

Adenoid cystic carcinoma (ACC) of salivary glands is characterized by a high rate of local recurrences, neurotropism and metastasis. ACC long-term survival rate is not promising. Thus, different chemotherapeutical approaches had been proposed for this neoplasm, including apoptosis induction by different drugs. This work evaluates the efficacy of Brefeldin-A (BFA), a potent apoptosis inducer, on ACC cultured cells (CAC2 cell line). CAC2 cells were treated with a 375 microM BFA solution in serum-free medium during 18 h. CAC2 cells grown in DMEM supplemented with 10% fetal bovine serum served as controls. Apoptotic cell recognition and counting were carried out through Hoechst staining. Transmission electron microscopy and immunofluorescence assessed the effect of BFA on CAC2 cells phenotype. Treated cultures showed a high apoptotic index presenting +/-30% of cells in evident apoptosis, when compared to controls. Apoptotic CAC2 cells also exhibited different alterations such as cytoplasmic vesicles formation and mitochondrial changes. Cultured ACC cells are strongly susceptible to apoptosis induction under BFA treatment, which may constitute a promising tool in further chemotherapeutical approaches.

Laminin-1 and SIKVAV a laminin-1-derived peptide, regulate the morphology and protease activity of a human salivary gland adenoid cystic carcinoma cell line.

In a previous paper, we demonstrated that laminin-1 and its derived peptide SIKVAV modulates the morphology of an adenoid cystic carcinoma cell line (CAC2 cells). Light microscopy of CAC2 cells grown in three-dimensional preparations of SIKVAV-enriched laminin-1 showed the presence of pseudocystic spaces. Pseudocysts are hallmarks of adenoid cystic carcinoma in vivo. We hypothesized that these pseudocystic spaces could be due to the protease-inducing/activating role of SIKVAV. Thus, we studied the presence of matrix metalloproteinases (MMPs) in CAC2 cells treated either by laminin-1 or by SIKVAV-enriched laminin-1. Immunohistochemistry and zymography suggested that SIKVAV enhanced the secretion of MMP-2 and MMP-9 in CAC2 cells. We propose that SIKVAV induces pseudocystic formation probably through the secretion of MMPs 2 and 9.

The effect of laminin and its peptide SIKVAV on a human salivary gland myoepithelioma cell line.

We have demonstrated that the basement membrane regulates the myoepithelioma. We are now studying the effect of laminin, a basement membrane protein, in the morphology of a cell line (M1) derived from human salivary gland plasmacytoid myoepithelioma. These cells were grown inside a three-dimensional preparation of laminin-1. Phenotype differences were assessed by light and transmission electron microscopy. In addition, we analysed the effect of a molecular domain of laminin-1, the peptide SIKVAV, on M1 cells. This peptide was chosen because it is effective in cell proliferation and differentiation. M1 cells grown inside laminin-1 were mostly plasmacytoid-like, while cells treated by SIKVAV showed light and electron microscopic features of typical plasmacytoid cells. This peptide also modulated smooth-muscle actin expression in M1 cells. We demonstrated that laminin-1 and its derived peptide SIKVAV morphoregulates myoepithelioma cells in culture.

Immunolocalization of bone extracellular matrix proteins (type I collagen, osteonectin and bone sialoprotein) in human dental pulp and cultured pulp cells.

AIM: To simultaneously analyse the expression of type I collagen, osteonectin and bone sialoprotein (BSP) in human dental pulp of different ages. METHODOLOGY: Cultured dental pulp fibroblasts (FP1 cell line), pulps from dental germs with incomplete root formation (n = 4) and pulps of erupted teeth with total root formation (n = 4) were used. Bone proteins were searched by immunohistochemistry and immunofluorescence using polyclonal antibodies and compared among the three groups assessed. RESULTS: Immunohistochemistry detected the three proteins in dental pulp tissue, as it labelled extracellular matrix, predentine and odontoblasts. The BSP label was weaker, when compared to both type I collagen and osteonectin. The presence of type I collagen was more evident in pulps from erupted teeth, when compared to germ dental pulps. On the other hand, a strong expression of osteonectin in germ dental pulps was observed. CONCLUSIONS: Regardless of the degree of maturation, dental pulps present type I collagen, osteonectin and BSP in the extracellular matrix (ECM) and in the odontoblastic layer. Thus, the results suggest that these proteins are related to the production and mineralization of dentine.

Analysis of epithelial-myoepithelial carcinoma based on the establishment of a novel cell line.

Epithelial-myoepithelial carcinoma of the salivary gland is a rare, low-grade, neoplasm, composed of ductal and myoepithelial cells. We present two novel cell lines, which have been characterised by immunofluorescence, derived from an epithelial-myoepithelial carcinoma of the parotid gland. A resected mass of the parotid gland was diagnosed as an epithelial-myoepithelial carcinoma by routine histological examination. Part of the specimen was labelled with a panel of antibodies confirming the tumour type. The other part was finely minced and the explants were incubated in DMEM supplemented with penicillin and streptomycin, at 37 degrees C in a humidified 5% CO(2) atmosphere. Two cell types were identified by immunofluorescence-a small cobblestone cell, positive for AE1/AE3 and p53, and a polyhedral cell, positive for vimentin, smooth muscle markers and S-100. Herein two cell lines are presented in order to open up possibilities of new studies and a discussion of the events that culminate in this bimodal neoplasm is also performed.

The effect of a reconstituted basement membrane (matrigel) on a human salivary gland myoepithelioma cell line.

We have already demonstrated that a reconstituted basement membrane (Matrigel) is a key modulator of morphogenetic changes and cytodifferentiation of pleomorphic adenoma cells in culture. Myoepithelioma is considered to be a neoplasm closely related to pleomorphic adenoma and should experience similar induction processes. Thus, the aim of this study was to investigate whether Matrigel would influence myoepithelioma cells. We used a cell line derived from a human salivary gland plasmacytoid myoepithelioma (M1 cells) grown in a three-dimensional preparation of Matrigel. Phenotype differences were assessed using conventional light microscopy technique (haematoxylin and eosin) and phase and differential interference contrast (Nomarski). Immunofluorescence was carried out to detect smooth-muscle actin, laminin and type-IV collagen. M1 cells exhibited all proteins studied, showing a myoepithelial differentiation. M1 cells grown inside Matrigel presented morphological changes and changes in smooth-muscle actin status. By growing M1 cells inside Matrigel, it was possible to reproduce the tumour architecture with no duct-like structures. Based on our findings, we suggest that myoepithelioma would be derived from a cell with a commitment to myoepithelial differentiation. We also suggest that the mechanical properties of the matrix environment will likely regulate smooth-muscle actin expression in myoepithelioma.

Effect of N-CAM on in vitro invasion of human adenoid cystic carcinoma cells.

Adenoid cystic carcinoma of salivary glands is characterised by aggressive behaviour, high rate of local recurrences, neurotropism and late metastasis. In a previous work we demonstrated that adenoid cystic carcinoma cultured cells (CAC2 cells) expressed N-CAM. It was suggested that this expression, modulated by extracellular matrix, would be correlated to cell movement. The aim of our study was to verify whether CAC2 cells presented invasion capacity. Moreover, we tested whether the neural adhesion molecule (N-CAM) would participate in this process. CAC2 cells were either previously treated, or not (control), with a monoclonal antibody against N-CAM. Invasion assays were carried out using a modified Boyden chamber (Transwell chamber). CAC2 cells (10(5)) were dispensed into Transwell upper chamber on the top of Matrigel coated filter. The cells that invaded the filters in the first 8 h were counted under light microscopy, yielding data for the invasion rates (%). Control CAC2 cells presented an invasion rate of 5.28+/-0.04%. The invasion rate raised to 6.53+/-0.2% when N-CAM was blocked with monoclonal antibody. N-CAM impaired the adenoid cystic carcinoma cell invasion in vitro. Therefore, we suggest an anti-invasive role for N-CAM in adenoid cystic carcinoma.

Verruciform xanthoma of the oral mucosa. Report of four cases and a review of the literature.

We present four new cases of verruciform xanthoma (VX) in the oral mucosa and review the literature. Clinical, histological, and immunohistochemical features of four new cases of VX were analysed together with cases found in a review of the literature. Expression of CD-68 was studied by immunohistochemistry. Only 162 cases were reported in the oral mucosa. Ninety were males (55.5%) and 72 were females (44.5%). Mean age was 44.9 years. The majority of cases occurred in masticatory mucosa (69.7%). Our cases exhibited papillary or verrucous proliferation of squamous epithelium associated with hyperparakeratosis and with numerous foamy cells confined to the lamina propria papillae. Foamy cells were positive to CD-68 antibody, showing a macrophagic nature. VX is a rare benign lesion, and is probably inflammatory. However, its aetiology and pathological mechanisms remain unknown.

Effect of a ceramic and a non-ceramic hydroxyapatite on cell growth and procollagen synthesis of cultured human gingival fibroblasts.

BACKGROUND: Ceramic hydroxyapatites and non-ceramic hydroxyapatites have been used extensively as alloplastic materials for bone reconstruction. However, different forms of hydroxyapatite induce different types of tissue response. METHODS: Human gingival fibroblasts (FMM1 cells) were used to analyze ceramic and non-ceramic hydroxyapatite biocompatibility. The cells were grown on surfaces covered either by collagen (control group), collagen plus ceramic hydroxyapatite, or collagen plus non-ceramic hydroxyapatite. Scanning electron microscopy, growth and cell viability curves, and procollagen immunoprecipitation were obtained. For the growth and viability curves, 10(4) cells were seeded on 60 mm dishes. Cells from each group were counted, in triplicate, at 1, 3, 4, 5, and 6 days after seeding using the Trypan blue dye exclusion assay. RESULTS: The cells grew in close contact with both types of hydroxyapatite particles. No differences were found in the amount of procollagen synthesis among any experimental group. The cultures treated with ceramic hydroxyapatite had a growth delay for the first 5 days. There was no difference in cell viability between the control group and the non-ceramic hydroxyapatite group. However, cultures treated with ceramic hydroxyapatite showed significantly lower viability percentages than the other groups. CONCLUSIONS: Hydroxyapatite supports cell growth and fibroblast metabolism including collagen production, and hence is biocompatible. Cell viability and structural studies showed that non-ceramic hydroxyapatite has relevant physical and biological properties as an implant material.

The role of basement membrane proteins on the expression of neural cell adhesion molecule (N-CAM) in an adenoid cystic carcinoma cell line.

We investigated the presence of neural cell adhesion molecule (N-CAM) in the adenoid cystic carcinoma cell line CAC2 using immunofluorescence microscopy. Additionally, we analysed the role of laminin and type IV collagen in N-CAM expression. We demonstrated that cultured adenoid cystic carcinoma cells express N-CAM. Control cells presented a scattered N-CAM expression on cell membrane, and type IV collagen had no effect in N-CAM distribution. CAC2 cells grown on laminin-coated coverslips expressed N-CAM concentrated on cell lamellipodia suggesting relationship with migratory activity. Our results showed that cultured adenoid cystic carcinoma cells express N-CAM and this expression is modulated by extracellular matrix.

Myofibroma of gingiva: report of a case with immunohistochemical and ultrastructural study.

Oral myofibroma is an uncommon, benign, solitary proliferation of myofibroblastic tissue. Few cases affecting maxillofacial region have been reported. We present a case of gingival myofibroma, diagnosed on clinical, histopathological, immunohistochemical, and ultrastructural basis.

Effect of spatial arrangement of the basement membrane on cultured pleomorphic adenoma cells. Study by immunocytochemistry and electron and confocal microscopy.

In a cell line from human pleomorphic adenoma (AP2 cells) we studied the response of these cells to basement membrane proteins. The culture was characterized as myoepithelial-like by transmission electron microscopy and immunocytochemistry. AP2 cells were grown in contact with a reconstituted basement membrane (Matrigel). Cells grown on Matrigel showed conspicuous phenotypic alterations, depending on how the substrate was applied. Cells grown on the top of Matrigel developed a dendritic phenotype, exhibiting thin, long and intercommunicating cytoplasmic extensions resembling normal myoepithelial cells. Cells grown inside Matrigel formed multi-layered clusters. Light, confocal and transmission electron microscopy showed that these clusters were formed by double-layered epithelioid cells delimiting luminal spaces. The cells facing the lumen were cuboidal, showing microvilli at the apical plasmalemmal and junctional complexes. The spatial arrangement of basement membrane is a key modulator of morphogenetic changes and cytodifferentiation of tumour myoepithelial cell lineage in culture.

Morphometric study of nucleolar organiser regions in ameloblastoma and basal cell carcinoma.

Ameloblastoma and basal cell carcinoma share histological similarities. Morphometric analysis of nucleolar organiser regions (NORs) from ameloblastoma and basal cell carcinoma (BCC) was carried out by silver (Ag) staining. Mean counts were lower in ameloblastoma (1.652 +/- 0.032) compared to those in BCC (2.354 +/- 0.054). Ameloblastoma presented one or two NORs per nucleus, in a narrow distribution (one to four NORs per nucleus). In contrast, BCC exhibited two or three NORs per nucleus, in a broad distribution (one to six NORs per nucleus). Perimeter and area measurements of AgNOR dots yielded significantly higher mean values for ameloblastoma. Our data suggest that most BCC cells are in mitosis, showing small and numerous NORs in each nucleus, while ameloblastoma cells are in interphase, showing one or two large NORs in each nucleus. Although ameloblastoma and BCC are neoplasms with similar growth patterns, they have cell populations with statistically significant differences in AgNOR patterns.

Expression of smooth-muscle actin in cultured cells from human plasmacytoid myoepithelioma.

OBJECTIVE: The definition of plasmacytoid myoepithelioma, a neoplasm exhibiting myoepithelial differentiation, has been recently questioned. To better understand the histogenesis of this neoplasm, we searched for myoepithelial markers in histologic sections of plasmacytoid myoepithelioma and in a cell line (M1) derived from this same neoplasm. STUDY DESIGN: Expression of vimentin, pan-keratin (AE-3) and smooth-muscle actin was studied by immunohistochemistry in paraffin-embedded tissue and by immunofluorescence in M1 cells. RESULTS: Plasmacytoid myoepithelioma tumor sections showed vimentin and AE-3 reactivity, but evidence of smooth-muscle actin was not seen. The cell line derived from this tumor also produced vimentin and cytokeratin. In addition, all cultured cells expressed smooth-muscle actin. CONCLUSION: We demonstrated that cells derived from a case of plasmacytoid myoepithelioma appear to show full myoepithelial differentiation in vitro. Thus, they are myoepithelial-like cells in nature. The lack of myogenous differentiation in vivo could be due to an inhibitory process mediated by the extracellular matrix.

Analysis of the interdependent localization of vimentin and microtubules in neoplastic myoepithelial cells.

Salivary gland neoplastic myoepithelial cells in culture form very thin cytoplasmic processes in which the vimentin network is well dispersed. These vimentin filaments can be individually visualized by immunofluorescence. In this study, we have analyzed the role of microtubules in the distension and organization of the vimentin filament network found in these cells. We find that vimentin filaments colocalize along microtubules; however, a significant number of filaments can also be found in microtubule-free domains. Additionally, vimentin filaments are absent from large domains of microtubule-rich domains. Treatment of neoplastic myepithelial cells with the microtubule inhibitor nocodazole did not cause any retraction of the distended vimentin network. This observation suggests that the structural integrity of microtubules is not important for the stability of the vimentin network. Combining procedures for transient disruption of vimentin filaments and microtubules we observed that, in the absence of microtubules, the vimentin network could reassemble in the perinuclear region but was unable to extend toward the cell periphery. The dispersion of vimentin filaments to the peripheral regions of the cytoplasm could only be observed upon microtubule reassembly. This indicates that microtubules are not required for the stability of the vimentin network, but the dispersion of vimentin filaments to the peripheral cytoplasm depends on active interactions with microtubules.

Microanatomic features of unilateral condylar hyperplasia.

Microanatomic features of unilateral condylar hyperplasia (UCH) are described. The articular surface exhibited clefts with surrounding elevations, and globules varying 0.5-2 microns in diameter. The articular zone presented giant coiled fibers, and the proliferative zone was composed of small round cells. The findings suggest that degenerative changes occur in UCH, both in adult and juvenile forms.

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: II. The role of microtubules in the organization of the cuticular plate.

The actin matrix of the cuticular plate, which supports the sensory stereocilia bundle, is coupled to the axial cytoskeleton of the hair cell through a well defined microtubule columnar framework. A collection of axial microtubules in a columnar organization penetrate deep into the dense actin matrix of the cuticular plate. Each microtubule displays at the end a 300-500 nm long fuzzy cap that enmeshes with the actin matrix of the cuticular plate. The microtubule associated proteins MAP-1A and MAP-1B were localized by confocal immunofluorescence to the point of microtubule insertion in the cuticular plate. These proteins are likely components of the microtubule capping structure and may mediate the interaction of the microtubules with the actin matrix. The structural interaction of the microtubules with the cuticular plate provides important mechanical coupling of the transduction apparatus to the axial cytoskeleton of the hair cell.

Caveat emptor: rank transform methods and interaction.

When distributional assumptions for analysis of variance are suspect, and nonparametric methods are unavailable, ecologists frequently employ rank transformation (RT) methods. The technique replaces observations by their ranks, which are then analysed using standard parametric tests. RT methods are widely recommended in statistics texts and in manuals for packages like SAS and IMSL. They are robust and powerful for the analysis of additive factorial designs. Recently, however, RT methods have been found to be grossly inappropriate for use with non-additive models. This severe limitation remains largely unreported outside of the theoretical statistics literature. Our goal is to explain this shortcoming of RT methods.