Expression and alterations of the PTEN / AKT / mTOR pathway in ameloblastomas.

OBJECTIVES: Recently, an allelic loss of phosphatase and tensin homologue (PTEN) was shown to occur in ameloblastomas. In carcinogenesis, loss of PTEN allows for overactivity of the phosphatidylinositol-3-kinase/protein kinase B (PI3K / AKT) pathway inducing an upregulation of mammalian-target of rapamycin (mTOR) and its downstream effector ribosomal-subunit-6 kinase (S6K); allowing for uncontrolled cell proliferation, apoptosis inhibition and cell cycle deregulation. METHODS: Thirty ameloblastomas and five dental follicles were studied, looking at the immunohistochemical expression of total PTEN and AKT, as well as their phosphorylated (p) active forms, and the downstream effector and indicator of mTOR activity p70 ribosomal-subunit-6 kinase (pS6K). Also assessed was the expression of extracellular-signal-regulated kinase (ERK), which cross talks with AKT. RESULTS: Total PTEN was absent in 33.3% of ameloblastomas, while its stabilized, phosphorylated(ser380 / thr382 / thr383) form was absent in 83.3% of tumors. In contrast, AKT was expressed in 83.3% of ameloblastomas, showing high expression of the p-thr(308)AKT and p-ser(473) AKT forms in 93.3% and 56.6% of cases, respectively. Further, the mTOR activated pS6K(ser240 / 244) was detected in 86.7% of ameloblastomas, while ERK was overexpressed in 70.0% of the cases. CONCLUSION: Immunohistochemical analysis of aberrant signaling in the PI3K/AKT/mTOR pathway in ameloblastomas may represent a valuable tool for elucidating pathogenesis, aggressiveness and selecting optimal therapeutics.

Survivin is a downstream target and effector of sulindac-sensitive oncogenic Stat3 signalling in head and neck cancer.

Sulindac exerts its antitumorigenic effects in oral squamous cell carcinoma (SCC) cells by modulating survivin in a Stat3-dependent manner. Immunohistochemistry was used to detect the protein levels of phosphorylated-tyrosine Stat3 (p-tyr Stat3) and survivin in SCC tissues. Western blot, reverse transcriptase polymerase chain reaction, Annexin-V and cell proliferation assays were used to determine p-tyr Stat3 and survivin protein and mRNA expression, and cell viability following treatment with cyclooxygenase (COX) inhibitors, Stat3 siRNA, or the forced expression of Stat3 or survivin. Immunohistochemical analysis revealed an overexpression of p-tyr Stat3 in T1 SCCs. The importance of constitutive Stat3 activation in tumourigenesis was confirmed by siRNA inhibition of Stat3, resulting in cell growth inhibition and apoptosis, via a downregulation of survivin mRNA and protein expression. The forced expression of survivin partially reversed these effects of Stat3 inhibition. Sulindac, but not other COX inhibitors, downregulated Stat3, which correlated to an inhibition of cell proliferation, survival and survivin expression. Transfection of constitutively active Stat3 restored survivin expression and partially rescued SCC cells from sulindac-induced antitumorigenic effects. These data indicate that survivin is a downstream target and effector of oncogenic Stat3 signalling in SCC, which is targeted by sulindac in a COX-2-independent manner.

Targeting the STAT pathway in head and neck cancer: recent advances and future prospects.

Head and neck cancer, the sixth most common type of cancer worldwide, is associated with a dismal prognosis that has minimally improved during the last few decades. Future advances in the treatment and prognosis of this fatal disease largely rely upon a better understanding of the molecular events that underlie tumor development and progression, allowing specific targeting of the involved molecules and pathways. In this context, recent efforts have revolved around a family of transcription factors known as STATs (signal transducers and activators of transcription). STAT proteins comprise a family of latent cytoplasmic transcription factors that become transiently activated in response to extracellular signals, leading to regulation of diverse physiological responses. There is compelling evidence that persistent activation of specific STAT molecules, especially Stat3 and Stat5, possesses oncogenic properties in a number of human cancers, including head and neck cancer. The presence of constitutively activated STAT molecules in cancer cells is mainly attributed to the dysregulation of upstream activating pathways and the aberration of negative regulatory mechanisms. The end result is induction of specific target genes that stimulate cell proliferation, prevent apoptosis, promote angiogenesis and facilitate tumor immune evasion. Therefore, targeting and disruption of oncogenic STAT signaling may theoretically be accomplished through various approaches, involving direct (e.g. interference with the various facets of STAT expression, activation or function) and indirect strategies (e.g. inhibition of upstream signaling events and enhancement or restoration of negative regulatory mechanisms). The availability of multiple potential targets for interruption of aberrant STAT signaling in cancer and the thus-far promising results have generated optimism for the clinical applicability of STAT targeting in head and neck cancer, which is the focus of this review.

Signal transducers and activators of transcription: insights into the molecular basis of oral cancer.

Recent efforts on developing more direct and effective targets for cancer therapy have revolved around a family of transcription factors known as STATs (signal transducers and activators of transcription). STAT proteins are latent cytoplasmic transcription factors that become activated in response to extracellular signaling proteins. STAT proteins have been convincingly reported to possess oncogenic properties in a plethora of human cancers, including oral and oropharyngeal cancer. Signal transduction pathways mediated by these oncogenic transcription factors and their regulation in oral cancer are the focus of this review.

Abrogation of IL-6-mediated JAK signalling by the cyclopentenone prostaglandin 15d-PGJ(2) in oral squamous carcinoma cells.

Cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) exerts antineoplastic effects on various types of human cancer. We recently showed that treatment with 15d-PGJ(2) induces apoptosis accompanied by downregulation of the oncogenic signal transducer and activator of transcription 3 (Stat3) signalling in human oral squamous cell carcinoma (SCC) cells. The current study examines the effects of 15d-PGJ(2) on the epidermal growth factor receptor (EGFR) and Janus Kinase (JAK)-mediated signalling pathways. Inhibition of Stat3 by 15d-PGJ(2) was abolished by exogenous stimulation with transforming growth factor alpha (TGF-alpha), but not interleukin 6 (IL-6), supporting a selective effect of 15d-PGJ(2) on IL-6-mediated signalling. Importantly, 15d-PGJ(2) selectively abrogated constitutive and IL-6-mediated JAK phosphorylation without affecting EGFR-activated levels. Moreover, the inhibitory effect of 15d-PGJ(2) on JAK signalling required the reactive alpha,beta-unsaturated carbon within the cyclopentenone ring. Targeting of JAK signalling using a specific JAK inhibitor also abolished Stat3 phosphorylation and resulted in apoptosis in oral SCC cells. Our findings provide the first evidence for 15d-PGJ(2)-mediated downregulation of constitutive and IL-6-induced JAK signalling in cancer and support that JAK inhibition and suppression of EGFR-independent Stat3 activation by 15d-PGJ(2) represent a promising approach for induction of apoptosis in oral SCC cells.

Cyclosporin A induces proliferation in human gingival fibroblasts via induction of transforming growth factor-beta1.

BACKGROUND: Cyclosporin A (CsA) is a widely used immunosuppressant that causes significant side effects including gingival overgrowth. The pathogenesis of this condition is not fully understood; however, recent studies show that CsA regulates the transcription of several cytokines including transforming growth factor-beta 1 (TGF-beta1). In this study, we evaluated the effects of CsA and TGF-beta1 on human normal gingival (NG) fibroblast proliferation, and explored a possible autocrine stimulation of TGF-beta1 as a cellular regulator of proliferation induced by CsA in NG fibroblasts. METHODS: NG fibroblast cell lines were incubated with increasing concentrations of CsA or TGF-beta1 and the proliferation index determined by automatic cell counting, BrdU incorporation, PCNA expression, and mitotic potential. To determine the effect of TGF-beta1 on the proliferation rate of NG fibroblasts under CsA treatment, NG fibroblast cultures were simultaneously treated with CsA and antisense oligonucleotides against the translation-start site of the TGF-beta1 mRNA. RESULTS: Treatment of NG fibroblasts with CsA or TGF-beta1 significantly stimulated the cell proliferation in a dose-dependent manner. Furthermore, neutralization of TGF-beta1 production in CsA-treated NG fibroblasts inhibited CsA's effect on NG fibroblast proliferation, demonstrating an autocrine stimulatory effect of TGF-beta1 in CsA-treated NG fibroblast proliferation. CONCLUSION: The results presented here suggest that CsA stimulatory induction of NG fibroblast proliferation is mediated via TGF-beta1 in an autocrine fashion.

Phenotypic switching of VEGF and collagen XVIII during hypoxia in head and neck squamous carcinoma cells.

The present study sought to determine the potential role of stress activated MAPK and phosphatidylinositol 3-kinase (PI3K) signaling pathways in mediating phenotypic switching between angiogenic and angiostatic elements among squamous cell carcinoma (SCC) cell lines. In particular, we investigated the effects of hypoxia and those of cobalt chloride (CoCl(2)), which mimics the hypoxic response including the production of reactive oxygen species, on such phenotypic shifts. The expression and production of collagen XVIII, and CBP2/Hsp47 provided a measure of an angiostatic phenotype, while vascular endothelial growth factor (VEGF) expression was used to assess potential angiogenic states. These studies revealed that hypoxia produced a slight up-regulation of collagen XVIII and CBP2/Hsp47 that was inhibited by the stress kinase inhibitor SB203580 but was unaffected by N-acetylcysteine (NAC). In addition, VEGF expression was increased following hypoxia and this effect was reversed with inhibition of by SB203580. Conversely, CoCl(2) significantly diminished the expression of both collagen XVIII and CBP2/Hsp47 and enhanced VEGF expression. These changes were reversed by the PI3K inhibitor wortmannin and by treating cells with NAC. These studies show that phenotypic switching between collagen XVIII and VEGF is controlled by stress activated kinases under hypoxia, and PI3K signaling pathways as well as reactive oxygen species (ROS) following CoCl(2) treatment. Furthermore, modulation of the angiogenic switch is most profound during Akt activation than during activation of stress activated kinases.

Expression of Sec61 alpha in F9 and P19 teratocarcinoma cells after retinoic acid treatment.

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (RE) membrane through a protein-conducting channel called the translocon. Sec61 alpha, a multispanning membrane translocon protein, has been implicated as essential for translocation of polypeptides chains into the cisterns of the ER. However, it is not known whether Sec61 alpha is ubiquitously expressed in collagen producing teratocarcinoma cells. Furthermore, the production, expression, and utilization of Sec61 alpha may depend on the cell differentiation stage. Stem cells from many cultured teratocarcinoma cell lines such as F9 and P19 cells are capable of differentiation in response to low retinoic acid concentrations. This differentiation of the tumorigenic stem cells results in tumorigenicity loss. For this study, mouse F9 and P19 teratocarcinoma cells were grown in culture medium treated with or without retinoic acid. Expression of Sec61 alpha was determined by reverse trancriptase polimerase chain reaction (RT-PCR). In untreated conditions, F9 cells expressed undetected Sec61 alpha amounts. It was also demonstrated that Sec61 alpha expression is stimulated in F9 cells after retinoic acid treatment for 72 hours. No changes were found in Sec61 alpha expression in P19 cells after retinoic acid treatment. These data indicate that the expression of Sec61 alpha is enhanced with retinoic acid induced differentiation of F9 teratocarcinoma cells.

Targeting of human squamous carcinomas by SPA470-doxorubicin immunoconjugates.

In a quest to identify a favorable target for head and neck cancers and squamous cell carcinoma, we sought to determine if Hsp47/CBP2 could be used as a target and whether the expression of this target was influenced by hypoxia. Moreover, we determined if doxorubicin (DOX) immunoconjugates directed against Hsp47/CBP2 that linked monoclonal antibodies (MAbs) to the 13-keto position of the drug possessed high cytotoxic drug activity and antibody-directed killing of antigen bearing tumor target cells. Experiments were performed using established cell lines of human oral squamous carcinoma cells (SCCs) (SCC-4, -9, -15 and -25) obtained from American Type Culture Collection (ATCC) (Manassas, VA). In addition, the UMB2 cell line is a spontaneous mutant of SCC-9 that does not express Hsp47/CBP2 was also used. Synthesis of the immunoconjugates was accomplished by thiolating the MAbs with 2 IT and reacting the MAbs with the DOX-hydrazone. The binding of MAb-DOX conjugates to SCC cells was determined by indirect immunofluorescence and analyzed using a Becton Dickinson FACS scan with Cell Quest software. Comparison of the cytotoxicity of DOX, MAb-DOX conjugates and MAb+DOX were determined using a limited dilution assay and colony survival assays during normoxia and hypoxia. These studies revealed that SCC cells treated with the SPA470-DOX conjugate for 2 h retained the original binding activity for targeted SCC cells and was significantly more potent that unconjugated DOX, DOX-hydrazone or equivalent MAb protein+DOX. Also, SPA47-DOX produced equal to and at lower concentrations greater cell killing than equivalent dose of free DOX. During hypoxia cells treated with SPA470-DOX demonstrated a small increase in colony survival and a diminishment in cytotoxicity. SPA470-DOX conjugates target SCC cells that express Hsp47/CBP2. The demonstration that SPA470-DOX is effective during hypoxia or conditions that mimic hypoxia presumes the further utility of SPA470-DOX in treating head and neck cancers.

Histomorphometric characteristics and expression of epidermal growth factor and its receptor by epithelial cells of normal gingiva and hereditary gingival fibromatosis.

OBJECTIVE: The objective of this study was to examine the histomorphometric features and evaluate the expression of epidermal growth factor (EGF) and transmembranic receptor (EGFr) and the proliferative potential of epithelial cells from normal and hereditary gingival fibromatosis (HGF) gingival tissues. BACKGROUND: EGF is a multifunctional cytokine with a variety of biological effects including stimulation of cell proliferation by binding to its specific EGFr. METHODS: Immunohistochemistry was performed to measure EGF and EGFr expression and the epithelial cell proliferation was determined by measuring proliferating cell nuclear antigen (PCNA). RESULTS: Histomorphometric evaluation indicated that in HGF the mean height of the epithelial papillae was higher compared to the normal gingiva (NG), whereas mean epithelial area and number of epithelial papillae were quite similar in both groups. The EGF and EGFr positive cells were observed in the basal, spinous and granular cell layers of both normal and HGF tissues, with a gradual reduction from the basal layer. Although the expressions of EGF and EGFr in the control group were significantly higher than those from HGF, in HGF the epithelial papilla tips showed increased number of proliferating cells and elevated expression of EGF and EGFr. There was a correlation between the proliferative potential of epithelial cells and the expression of EGF or EGFr only in the epithelial papilla tips of HGF gingiva. CONCLUSION: Our data suggest that EGF and EGFr in the oral epithelium of HGF gingiva may stimulate epithelial cell proliferation, with the resultant apical migration of the oral epithelium and formation of the slender deep epithelial papillae; however, without hyperplastic alterations.

Expression of Sec61alpha in F9 and P19 teratocarcinoma cells after retinoic acid treatment

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (RE) membrane through a protein-conducting channel called the translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as essential for translocation of polypeptides chains into the cisterns of the ER. However, it is not known whether Sec61alpha is ubiquitously expressed in collagen producing teratocarcinoma cells. Furthermore, the production, expression, and utilization of Sec61alpha may depend on the cell differentiation stage. Stem cells from many cultured teratocarcinoma cell lines such as F9 and P19 cells are capable of differentiation in response to low retinoic acid concentrations. This differentiation of the tumorigenic stem cells results in tumorigenicity loss. For this study, mouse F9 and P19 teratocarcinoma cells were grown in culture medium treated with or without retinoic acid. Expression of Sec61alpha was determined by reverse trancriptase polimerase chain reaction (RT-PCR). In untreated conditions, F9 cells expressed undetected Sec61alpha amounts. It was also demonstrated that Sec61alpha expression is stimulated in F9 cells after retinoic acid treatment for 72 hours. No changes were found in Sec61alpha expression in P19 cells after retinoic acid treatment. These data indicate that the expression of Sec61alpha is enhanced with retinoic acid induced differentiation of F9 teratocarcinoma cells

Effect of transforming growth factor-beta1, interleukin-6, and interferon-gamma on the expression of type I collagen, heat shock protein 47, matrix metalloproteinase (MMP)-1 and MMP-2 by fibroblasts from normal gingiva and hereditary gingival fibromatosis.

BACKGROUND: Increased collagen and extracellular matrix deposition within the gingiva is the main characteristic feature of hereditary gingival fibromatosis (HGF). To date, it is not well established if these events are a consequence of alterations in the collagen and other extracellular matrix molecules synthesis or disturbances in the homeostatic equilibrium between synthesis and degradation of extracellular matrix molecules. Cytokines are important regulators of expression of the profibrogenic genes, including type I collagen and its molecular chaperone heat shock protein (Hsp)47 and proteolytic enzymes degrading extracellular matrix such as matrix metalloproteinases-1 and -2 (MMP-1 and MMP-2). METHODS: In this study, we analyzed the expression and production of type I collagen, Hsp47, MMP-1, and MMP-2 in normal gingiva (NG) and HGF fibroblasts, and investigated the effects of transforming growth factor-beta1 (TGF-beta1), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) on the expression of these genes by NG and HGF fibroblasts. RESULTS: Our results obtained from semi-quantitative reverse transcription-polymerase chain reactions (RT-PCR), Western blots, enzyme-linked immunosorbent assays (ELISA), and enzymographies clearly demonstrated that the expression and production of type I collagen and Hsp47 were significantly higher in fibroblasts from HGF than from NG, whereas MMP-1 and MMP-2 expression and production were lower in fibroblasts from HGF patients. Addition of TGF-beta1 and IL-6, which are produced in greater amounts by HGF fibroblasts, promoted an increase in type I collagen and Hsp47 and a decrease in MMP-1 and MMP-2 expression. IFN-gamma reduced both type I collagen and Hsp47 expression, whereas it had a slight effect on the expression of MMP-1 and MMP-2. CONCLUSION: These patterns of expression and production suggest that enhanced TGF-beta1 and IL-6 production simultaneously increase the synthesis and reduce the proteolytic activities of fibroblasts from patients with HGF, which may favor the accumulation of extracellular matrix observed in patients with this condition.

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment.

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37 degrees C and 43 degrees C (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37ºC and 43ºC (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV

Expression of matrix metalloproteinases in cyclosporin-treated gingival fibroblasts is regulated by transforming growth factor (TGF)-beta1 autocrine stimulation.

BACKGROUND: Gingival overgrowth is a common side effect following the administration of cyclosporin A (CsA). The pathogenesis of this condition is not fully understood; however, recent studies show that CsA regulates the transcription of several cytokines including transforming growth factor-beta1 (TGF-beta1). The aim of this study was to investigate the potential role of TGF-beta1 in the pathogenesis of CsA-induced gingival overgrowth, exploring a possible autocrine stimulation of TGF-beta1 as a cellular regulator of synthesis of matrix metalloproteinases (MMPs) and its tissue inhibitors (TIMPs). METHODS: Gingival fibroblasts from human normal gingiva were incubated with increasing concentrations of CsA, cultured for 24 hours, and the expression and production of TGF-beta1 determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. MMP and TIMP mRNA expression levels were also analyzed by RT-PCR. To determine the effect of TGF-beta1 on the expression of MMP and TIMP by human gingival fibroblasts under CsA treatment, human gingival fibroblast cultures were treated with sense oligonucleotides (SON) or antisense oligonucleotides (AON). RESULTS: CsA simultaneously stimulated TGF-beta1 expression and production and inhibited expression of MMP-1 and MMP-2 by human gingival fibroblasts, whereas CsA has a slight effect on TIMP-1 and TIMP-2 expression. AON reduced TGF-beta1 production as demonstrated by ELISA, whereas TGF-beta1 mRNA expression levels were not significantly modified. The inhibition of TGF-beta1 production by AON modulated MMP expression, demonstrating the autocrine inhibitory effect of TGF-beta1 in CsA-treated human gingival fibroblasts. CONCLUSIONS: The data presented here suggest that TGF-beta1 in an autocrine fashion may contribute to a reduction of proteolytic activity of human gingival fibroblasts in CsA-induced gingival overgrowth, which favors the accumulation of extracellular matrix.

15-PGJ2, but not thiazolidinediones, inhibits cell growth, induces apoptosis, and causes downregulation of Stat3 in human oral SCCa cells.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been linked to induction of differentiation, cell growth inhibition and apoptosis in several types of human cancer. However, the possible effects of PPARgamma agonists on human oral squamous cell carcinoma have not yet been reported. In this study, treatment with 15-deoxy-Delta(12,14)-PGJ(2) (15-PGJ(2)), a natural PPARgamma ligand, induced a significant reduction of oral squamous cell carcinoma cell growth, which was mainly attributed to upregulation of apoptosis. Interestingly, rosiglitazone and ciglitazone, two members of the thiazolidinedione family of PPARgamma activators, did not exert a growth inhibitory effect. Given the critical role that the oncogene signal transducer and activator of transcription 3 (Stat3) plays in head and neck carcinogenesis, its potential regulation by PPARgamma ligands was also examined. Treatment of oral squamous cell carcinoma cells with 15-PGJ(2) induced an initial reduction and eventual elimination of both phosphorylated and unphosphorylated Stat3 protein levels. In contrast, other PPARgamma did not induce similar effects. Our results provide the first evidence of significant antineoplastic effects of 15-PGJ(2) on human oral squamous cell carcinoma cells, which may be related to downmodulation of Stat3 and are at least partly mediated through PPARgamma-independent events.

Expression of Matrix Metalloproteinases in Cyclosporin-Treated Gingival Fibroblasts Is Regulated by Transforming Growth Factor (TGF)-ß1 Autocrine Stimulation / x

Background: Gingival overgrowth is a common side effect following the administration of cyclosporin A (CsA). The pathogenesis of this condition is not fully understood; however, recent studies show that CsA regulates the transcription of several cytokines including transforming growth factor-ß1 (TGF-ß1). The aim of this study was to investigate the potential role of TGF-ß1 in the pathogenesis of CsA-induced gingival overgrowth, exploring a possible autocrine stimulation of TGF-ß1 as a cellular regulator of synthesis of matrix metalloproteinases (MMPs) and its tissue inhibitors (TIMPs). Methods: Gingival fibroblasts from human normal gingiva were incubated with increasing concentrations of CsA, cultured for 24 hours, and the expression and production of TGF-ß1 determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. MMP and TIMP mRNA expression levels were also analyzed by RT-PCR. To determine the effect of TGF-ß1 on the expression of MMP and TIMP by human gingival fibroblasts under CsA treatment, human gingival fibroblast cultures were treated with sense oligonucleotides (SON) or anti-sense oligonucleotides (AON). Results: CsA simultaneously stimulated TGF-ß1 expression and production and inhibited expression of MMP-1 and MMP-2 by human gingival fibroblasts, whereas CsA has a slight effect on TIMP-1 and TIMP-2 expression. AON reduced TGF-ß1 production as demonstrated by ELISA, whereas TGF-ß1 mRNA expression levels were not significantly modified. The inhibition of TGF-ß1 production by AON modulated MMP expression, demonstrating the autocrine inhibitory effect of TGF-ß1 in CsA-treated human gingival fibroblasts. Conclusions: The data presented here suggest that TGF-ß1 in an autocrine fashion may contribute to a reduction of proteolytic activity of human gingival fibroblasts in CsA-induced gingival overgrowth, which favors the accumulation of extra-cellular matrix

Ameloblastoma in children.

PURPOSE: The purpose of this report was 1) to report the experience of the University of Maryland, Department of Oral and Maxillofacial Surgery (OMS Department) in the treatment of ameloblastoma in children and 2) to review the world literature on the treatment of ameloblastoma in children from 1970 to 2001. METHODS AND MATERIALS: This study first reviews the experience of the OMS Department of the University of Maryland with ameloblastomas in children and then reviews the literature on this subject. The first part of the study was undertaken by a retrospective chart review of all patients with a diagnosis of ameloblastoma in the OMS Department between May 1991 and December 1999. The literature on ameloblastoma in Western societies and Africa was separately reviewed from 1970 through 2001. Reports earlier than 1970 were not reviewed, as the histologic diagnosis of ameloblastoma was not well defined before that period. RESULTS: In the Maryland series, 11 patients under the age of 20 years with ameloblastoma were treated. Eight patients were seen primarily, and 3 presented with recurrent lesions. The average age was 15.5 years; 5 of 11 patients were black, and 9 of 11 tumors were unicystic ameloblastomas. The literature review showed 85 children in the Western reports and 77 reported from Africa. The average ages were 14.3 and 14.7 years, respectively, but unicystic ameloblastomas accounted for 76.5% of the Western and only 19.5% of the African children, with an increased frequency of occurrence in the mandibular symphisis in African (44.2%) versus Western (5.8%) patients. Analysis of recurrence after enucleation of unicystic ameloblastomas in 20 children followed at least 5 years or until recurrence showed a recurrence of 40%. CONCLUSIONS: Ameloblastomas in children differ from adults, with a higher percentage of unicystic tumors. African children appear to resemble the adult pattern. Although enucleation has been claimed to give acceptable recurrence rates in unicystic ameloblastoma, there are no large series with long follow-up in children. The histologic pattern that exhibits mural invasion in unicystic ameloblastoma suggests that more aggressive surgery is necessary.

Testosterone stimulates proliferation and inhibits interleukin-6 production of normal and hereditary gingival fibromatosis fibroblasts.

Hereditary gingival fibromatosis (HGF) is a rare oral condition characterized by a slow and progressive enlargement of the gingiva, involving both the maxilla and mandible. In vitro, HGF fibroblasts demonstrate a proliferative index significantly higher than fibroblasts from normal gingiva (NG). The objective of this study was to determine the effect of dihydrotestosterone on the proliferation of gingival fibroblasts derived from patients with HGF (n = 4) and from four healthy individuals. Additionally, we analyzed the effect of dihydrotestosterone on interleukin-6 (IL-6) production and determined the expression levels of androgen receptors in NG and HGF fibroblasts. Gingival fibroblasts from NG and HGF were incubated with increasing concentrations of dihydrotestosterone with or without androgen blockers, and cultured for 24 h, and the proliferation index was determined by automated cell counter. IL-6 production, in this system, was quantified using a "capture" enzyme-linked immunosorbent assay (ELISA). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to measure the mRNA expression of androgen receptors. The results indicated that dihydrotestosterone simultaneously downregulates the production of IL-6 and upregulates the cell proliferation. Finasteride and cyprosterone acetate, two anti-androgens, partially reversed these effects. Androgen receptor mRNA expression was identified in both NG and HGF fibroblasts; however, the levels in NG were higher than those observed in HGF. These results show that testosterone coordinates the proliferation and production of IL-6 of normal and HGF fibroblasts.

Neurofibromatosis type 1: a clinicopathological study of the orofacial manifestations in 6 pediatric patients.

Neurofibromatosis type 1 (NF1) is a relatively frequent mucocutaneous syndrome, which is transmitted as an autosomal dominant trait or which may represent neomutation. It is characterized by a variety of clinical manifestations, including multiple neurofibromas that are associated with a high risk of sarcomatous transformation. The aim of this report was to elucidate the orofacial manifestations observed in 6 pediatric patients (between 4 and 15 years of age) diagnosed with NF1. Physical, clinical, radiological, histological, and immunohistochemical studies were performed. Orofacial lesions were observed in all studied patients, located either in the soft tissues (4 cases) or centrally in the jaws (2 cases). All cases showed facial asymmetry, one of them exhibiting marked facial hemihypertrophy. All cases with soft tissue involvement were plexiform neurofibromas, while the intraosseous cases were diagnosed as solitary neurofibromas. Knowledge of the variability of presentation of orofacial soft tissue and bone manifestations of NF1 in children is necessary for prompt diagnosis.