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The pathogenesis of oral cancer is a complex and multifactorial process that requires a deep understanding of the underlying mechanisms involved in the development and progress of malignancy. The ever-improving comprehension of the diverse molecular characteristics of cancer, the genetic and epigenetic alterations of tumor cells, and the complex signaling pathways that are activated and frequently cross talk open up promising horizons for the discovery and application of diagnostic molecular markers and set the basis for an era of individualized management of the molecular defects underlying and governing oral premalignancy and cancer. The purpose of this article is to review the key molecular concepts that are implicated in oral carcinogenesis, especially focusing on oral squamous cell carcinoma, and to review selected biomarkers that play a substantial role in controlling the so-called "hallmarks of cancer," with special reference to recent advances that shed light on their deregulation during the different steps of oral cancer development and progression.

Identification and management of potentially premalignant oral epithelial lesions (PPOELs) at highest risk of malignant transformation holds great promise for successful secondary prevention of oral squamous cell carcinoma, potentially reducing oral cancer morbidity and mortality. However, to date, neither clinical nor histopathologic validated risk predictors that can reliably predict which PPOELs will definitively progress to malignancy have been identified. In addition, the management of PPOELs remains a major challenge. Arguably, progress in the prevention and treatment of oral premalignancy and cancer will require improved understanding of the underlying molecular mechanisms, facilitating the discovery of diagnostic, prognostic, and predictive markers, as well as the identification of novel targeted therapeutics. This review provides a synopsis of the molecular biomarkers that have been studied in PPOELs and have been correlated with the presence and grade of dysplasia and/or their propensity to undergo malignant transformation to oral squamous cell carcinoma. The emphasis is on highlighting new emerging research fields, particularly epigenetic events, including methylation and micro-RNA regulation. Several promising biomarkers are highlighted. Current limitations and challenges are discussed. Recommendations for future focused research areas, to validate and promote clinically useful applications, are offered.

In recent years, signal transducers and activators of transcription (STAT) proteins have been recognized as cytoplasmic transcription factors that mediate extracellular signaling to the nucleus controlling fundamental functions, such as cell proliferation, apoptosis, differentiation, immune responses and angiogenesis. Among them, STAT3 is a major player, aberrant activation of which is involved in several diseases, including cancer. Among other upstream regulators, IL-6/Jak signaling can activate STAT3 and its role appears to be critical in various types of cancer. Although STAT3 has been traditionally recognized as amoncogene, more recently the dual role of STAT3 in cancer, either tumor inductive or suppressive, has been appreciated. The importance and differential effect of STAT3 on tyrosine or serine residues are also a matter of continuing debate. Interestingly, recent findings suggesting that STAT3 plays an important role in cancer stem cell regulation have gained significant attention. This review summarizes current literature focusing on the significance of STAT3 in several diseases as well as in cancer. Understanding the complexity of STAT3 function has the potential to elucidate important molecular aspects of cancer with significant therapeutic implications.

Orbital floor fractures are a serious consequence of craniofacial trauma and account for#x223C;60%-70% of all orbital fractures. Unfortunately, the body's natural response to orbital floor defects generally may not restore proper function and facial aesthetics, which is complicated by the thin bone and adjacent sinuses. Current clinical treatments include alloplastic implants and autologous grafts; however, each has associated disadvantages and sequelae. This review has outlined necessary components for a successful tissue-engineered construct for orbital floor repair. In addition, current successes and progress in the literature specific to orbital floors and craniofacial research have been reviewed. Finally, challenges and future directions have been described.

Objective Recent identification of altered molecular signaling pathways in neoplasia has begun to elucidate mechanisms of oncogenesis, differentiation, and tumor progression, and to suggest plausible nonsurgical considerations for treatment. Here we review the sonic hedgehog (SHH) and PI3K/Akt/mTOR signaling pathways, their role in ameloblastoma, a locally aggressive odontogenic tumor, and evidence for consideration of therapeutic approaches that target these molecular pathways. In so doing, some of the gaps will be revealed that may impel investigations and translate to patient care, helping to minimize or eliminate the need for extensive surgery. Study design This is a comprehensive review of the literature regarding alterations in signaling mechanisms associated with ameloblastomas. In addition, this review attempts to explore and discuss possible inhibitors to these pathways that may have utility in treating ameloblastoma. Results The expression of SHH signaling molecules in ameloblastomas at the mRNA and protein levels has intimated that these molecules may play a role in cell proliferation of these tumors. Immunohistochemical analysis has revealed aberrant signaling in the PI3K/Akt/mTOR pathway in ameloblastomas and appears to be a valuable tool for elucidating pathogenesis and aggressiveness, and selecting optimal therapeutics. Conclusion The understanding of altered pathways in ameloblastoma may soon provide nonsurgical options for the treatment of this condition. The demonstration of cross talk in SHH signal transduction with PI3K signaling through Akt has shown that these pathways converge to control the Gli transcription factors. Thus, tumors that entirely depend on active SHH signaling for survival/growth and maintenance may well be susceptible targets for combined chemotherapy with SHH-specific inhibitors together with PI3K, Akt, or mTOR blocking agents. Some of these inhibitors could be used locally, thereby minimizing major systemic effects.

We have incorporated hydroxyapatite nanoparticles within cyclic acetal hydrogels to create nanocomposites that can be used to repair surgically created orbital floor defects in a rabbit animal model. Nanosized hydroxyapatite particles may improve tissue engineering scaffold properties because they have similar length scale of many cellular and molecular components and therefore can enhance cellular adhesion and migration. We hypothesize that inclusion of nanosized hydroxyapatite particles (20-70 nm) within cyclic acetal hydrogels would support bone defect repair. The objectives of our study include (1) characterization of nanocomposites in vitro, (2) investigation of tissue response and capsule tissue surrounding nanocomposites in vivo, and (3) investigation of the potential of nanocomposites to facilitate bone formation at 7- and 28-day time points in vivo. Experimental nanocomposite groups consisted of 0, 10, and 50 ng/mL nanosized hydroxyapatite. In vitro results indicated uniform dispersion of nanoparticles within nanocomposites and increased compressive moduli of nanocomposites with increase in nanoparticle concentration and bone marrow stromal cell viability within nanocomposites. In vivo results at day 7 indicated a tissue response of mild to increased inflammatory cells and presence of immature fibrous tissue. At day 28, tissue response consisted of mild inflammatory response and mature tissue. Quantitative results at day 7 indicated no difference in total bone percentage area between groups. The results also indicated that the tissue capsule surrounding the 0, 10, and 50 ng group implants had no clear organization. Quantitative results at day 28 indicated that the tissue capsule surrounding the 0, 10, and 50 ng group implants was an organized layer and the bone percentage for the 50 ng group was significantly higher than that of the remaining groups. Initial results indicated that our nanocomposites initiate a positive in vivo response in terms of bone growth. However, the percentage of bone area compared with the total area was low at both time points. Thus, in our study, even after addition of nanoparticles to cyclic acetal hydrogels, their biocompatible properties were maintained. On the other hand, addition of nanoparticles to cyclic acetal hydrogels did not lead to complete restoration of orbital floor defects.

Constitutive activation of the signal transducer and activator of transcription 3 (Stat3) has been detected in various human cancers and has been linked to tumor development and progression. Oncogenic Stat3 signaling results in induction of specific target genes, among which survivin is implicated in the proliferation and survival of cancer cells. Targeting of Stat3 constitutive expression by the nonsteroidal antiinflammatory drug (NSAID) sulindac has been demonstrated to exert antineoplastic effects in oral squamous cell carcinoma cells in vitro and in vivo.The expression and functional role of Stat3 and survivin was evaluated in 2 salivary gland adenocarcinoma cell lines (HSY and HSG). In addition, the effects of the NSAID sulindac and other cyclooxygenase (COX) inhibitors on Stat3 and survivin expression and on cell proliferation and apoptosis of HSY and HSG cells were analyzed.Messenger RNA and protein expression of Stat3 and survivin was detected in HSY and HSG cell lines. Treatment of these cells with siRNA against Stat3 or survivin inhibited cell proliferation and induced apoptosis. Moreover, Stat3 siRNA treatment down-regulated the protein and mRNA expression of survivin, and survivin forced expression partially reversed the antineoplastic effects of Stat3 siRNA treatment. Treatment of HSY and HSG cells with the NSAID sulindac, but not other COX inhibitors, induced significant decreases in cell proliferation and increases in apoptosis, accompanied by down-regulation of Stat3 and survivin expression. In contrast, survivin forced expression or transfection with constitutively active Stat3 attenuated the effects of sulindac on cell growth and apoptosis.Taken together, these data support the importance of the constitutive Stat3 signaling for growth and survival of salivary gland cancer cells through the induction of survivin. Inhibition of the oncogenic Stat3-survivin pathway in these cells can be achieved by selective targeting techniques or treatment with the NSAID sulindac and holds promise for the treatment of salivary gland cancer.

Perisinusoidal hepatic stellate cells (HSC) are the principal fibrogenic cells in the liver. In animal models, HSC apoptosis is the predominant clearance mechanism of activated HSC, although data evaluating whether the same processes occur in humans are limited. We conducted a cross-sectional study to evaluate the association between HSC apoptosis and fibrosis stage in subjects with chronic hepatitis C virus (HCV) infection (n = 44) and HCV-negative controls with normal liver histology (n = 9). We used immunohistochemical techniques to identify activated (alpha-smooth muscle actin+), proliferative (Ki-67+) and apoptotic (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end-labelling+) HSC in liver biopsy specimens from all subjects. The same pathologist enumerated positive cells per high-power field (HPF, x 200) in 20 periportal/lobular areas. HSC apoptosis was decreased in HCV-positive subjects compared with controls (median 0.4, range 0.0-3.1 vs 1.1, 0.2-3.5 cells/HPF, P = 0.02). Among HCV-positive subjects, HSC apoptosis was decreased in those with moderate to advanced fibrosis (P = 0.04) compared with those with mild fibrosis. By multivariate analysis, HSC apoptosis decreased by an average of 0.14 cells/HPF (95% confidence interval 0.01-0.28 cells/HPF) per increase in fibrosis stage (P = 0.04). While the number of activated and proliferative HSC was significantly increased in HCV-infected subjects compared with that in uninfected controls, the numbers of these cells did not differ between HCV-infected subjects with mild vs moderate/advanced fibrosis. In conclusion, the number of apoptotic HSC was significantly decreased in HCV-infected subjects with advanced fibrosis. In chronic HCV infection, inhibition of HSC apoptosis may be one mechanism by which fibrosis progresses.

Many systems have been proposed for the encapsulation of bone marrow stromal cells (BMSCs) within degradable hydrogels. Here, we use a novel cyclic acetal-based biomaterial formed from 5-ethyl-5-(hydroxymethyl)-beta,beta-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD) and poly(ethylene glycol) diacrylate (PEGDA). A cyclic acetal-based hydrogel may be preferred as cyclic acetals hydrolytically degraded into diols and carbonyls as primary degradation products, which may not affect local acidity, unlike other widely investigated polymers. The EHD monomer and PEGDA polymer may be fabricated into a EH-PEG hydrogel by radical polymerization initiated by the ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) system. The objective of this work is to determine whether the components utilized in the fabrication of EH-PEG hydrogels as well as the EH-PEG hydrogels permit BMSC viability, metabolic activity, and osteodifferentiation. Cell viability and metabolic activity were assessed after 30 min, 1 h, and 3 h of exposure to pertinent concentrations of the initiator system (10-20 mM). Osteodifferentiation was assessed by alkaline phosphatase and osteocalcin expression after a short exposure to the initiator system to simulate the encapsulation process. Lastly, cell viability was assessed immediately after encapsulation and after 7 days of culture within the EH-PEG hydrogels. Results indicate that the metabolic activity and viability of BMSCs are minimally affected, and that osteodifferentiation is not significantly affected by the APS-TEMED initiator system. Also, encapsulated BMSCs maintained viability within EH-PEG hydrogels for 7 days. This work demonstrates that the EH-PEG hydrogel is a viable option for the encapsulation and osteodifferentiation of BMSCs.

Purpose Clefts of the lip and palate are the most prevalent congenital craniofacial birth defect in humans. The developing field of tissue engineering is considered for the management of clefts of the lip and palate. Materials and Methods A review of the literature was carried out by using electronic databases (such as PubMed and ISI Web of Science) to search topics including “cleft palate,” “tissue engineering,” “bone engineering,” “palate engineering,” and “alveolar bone grafting.” To reflect current practice and research, these searches were limited primarily to articles published after the year 2000. Results Current approaches for the treatment of clefts of the lip and palate include surgery and bone grafts; however, there are limitations associated with these therapies. Tissue engineering strategies, particularly alveolar bone engineering and soft tissue engineering, may provide clinicians with new alternatives. The application of these emerging technologies to a pediatric population must be well considered. Conclusions A tissue engineering approach may be a useful alternative for the treatment of cleft palates as it mitigates the concerns of donor site morbidity as well as provides additional options including scaffold implantation and growth factor delivery.

Tonsil epithelium has been implicated in human immunodeficiency virus (HIV) pathogenesis, but its role in oral transmission remains controversial. To study characteristics of this tissue, which may influence susceptibility or resistance to HIV, we performed microarray analysis of the tonsil epithelium. Our data revealed that genes related to immune functions such as antibody production and antigen processing were increasingly expressed in tonsil compared with the epithelium of another oropharyngeal site, the gingival epithelium. Importantly, tonsil epithelium highly expressed genes associated with HIV entrapment and/or transmission, including the HIV co-receptor CXCR4 and the potential HIV-binding molecules FcRgammaIII, complement receptor 2, and various complement components. Immunohistochemical staining confirmed the increased presence of CXCR4 in the tonsil epithelium compared with multiple oral epithelial sites, particularly in basal and parabasal layers. This increased expression of molecules involved in viral recognition, binding, and entry may favor virus-epithelium interactions in an environment with reduced innate antiviral mechanisms. Specifically, secretory leukocyte protease inhibitor, an innate molecule with anti-HIV activity, was minimal in the tonsil epithelium, in contrast to oral mucosa. Collectively, our data suggest that increased expression of molecules associated with HIV binding and entry coupled with decreased innate antiviral factors may render the tonsil a potential site for oral transmission.

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.

Purpose Lymph node metastasis from oral squamous cell carcinoma (SCC) correlates with a poor prognosis. Therefore, accurate assessment of lymph node status is crucial in treatment planning. Furthermore, prediction of delayed neck metastasis (DNM), especially in early stage tumors with a clinically negative (N0) neck, will determine the need for neck dissection or irradiation. In this study, we assess various clinical, histopathological and lymphangiogenic parameters in early stage oral SCC and their association with DNM. Materials and Methods Clinical, histological, and immunohistochemical analyses were undertaken for 29 patients with T1N0M0 or T2N0M0 oral SCC affecting the tongue or floor of mouth and correlated with the development of DNM. Results Tumor thickness, nuclear pleomorphism, pattern of invasion, and immunohistochemical expression of the lymphangiogenesis-associated molecules VEGFR-3 and VEGF-C were associated with DNM. Conclusions Analysis of these parameters may help to identify patients who would benefit from a neck dissection or irradiation by predicting the likelihood of lymph node metastasis.

Background. Proliferative myositis is a rare, benign, reactive intramuscular lesion of fibroblastic/myofibroblastic origin; an identical lesion in a subcutaneous or fascial location is referred to as proliferative fasciitis. The rapid growth rate and unusual histopathologic features have frequently been mistaken for a malignant process and have promoted unnecessary invasive procedures. Here we present only the third oral case of proliferative myositis, arising from the tongue of a 65-year-old man. Methods and Results. Histologically, the resected lesion was composed of numerous fibroblastic or myofibroblastic spindle cells and variable numbers of large ganglion-like cells infiltrating between and around muscle fascicles, resembling a “checkerboard” configuration. A demographic profile of proliferative myositis of the head and neck is also provided, compiled from 19 patients culled from an English-language literature review and this report. Conclusions. Incisional biopsy or fine-needle aspiration biopsy of proliferative myositis of the head and neck should lead to spontaneous resolution and is, therefore, sufficient to render the diagnosis and to provide conservative treatment. Recurrence is extremely rare. © 2006 Wiley Periodicals, Inc. Head Neck, 2007

Inflammatory myofibroblastic tumor (IMT) is a controversial lesion composed of myofibroblasts, accompanied by varying numbers of inflammatory cells. Various pathogenetic factors have been proposed (ie, reactive, infectious, autoimmune, and neoplastic) but the etiology of most IMTs remains unknown. Here we review the literature of oral IMTs, detailing the demographic profile of these rare lesions. Moreover, we present an unusual case of IMT arising from the mandibular alveolar mucosa of an 82-year-old female. Microscopic examination revealed plump spindle cells set in a myxoid vascular stroma admixed with inflammatory cells. Numerous large ganglion cell-like cells were seen, some exhibiting emperipolesis of neutrophils. Ultrastructurally, prominent myofibroblasts with abundant rough endoplasmic reticulum were noted. Tumor cells were immunoreactive for vimentin, smooth muscle actin, and KP1 (CD68), and negative for desmin, S-100, and EBV-LMP. The lesion was excised without margins and the patient has manifested no evidence of disease at an 18-month recall. In an attempt to further delineate the potential neoplastic nature of this lesion, we assessed the immunohistochemical expression of various markers that have been linked to neoplastic transformation. The recorded positivity for ALK, p53, MDM2, CDK4, pRb, and Ki-67, despite the absence of bcl-2 reactivity, strongly favors the neoplastic origin of the studied tumor.

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.

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.

Antineoplastic properties of cyclopentenone 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) involve peroxisome proliferator-activated receptor gamma (PPARgamma) dependent and independent mechanisms. We recently reported that 15d-PGJ2 inhibits cell growth and induces apoptosis in human oral squamous cell carcinoma (SCC) partly independent of PPARgamma activation. Given the importance of epidermal growth factor receptor (EGFR) as a therapeutic target in head and neck SCC, we addressed the effects of 15d-PGJ2 on EGFR expression. 15d-PGJ2, but not other PPARgamma ligands, abrogated EGFR protein expression in oral SCC cells. 15d-PGJ2 also decreased EGFR mRNA, indicating downmodulation at the transcriptional level. Moreover, treatment with 9,10-dihydro-15d-PGJ2, a 15d-PGJ2 analog lacking the reactive carbonyl group, failed to effect EGFR expression. These findings provide evidence for EGFR downregulation in oral SCC cells through a novel anticancer effect of 15d-PGJ2 that is attributed to the reactive cyclopentenone ring system.

Squamous carcinomas of the head and neck (HNSCC) represent the sixth most common cancer among men worldwide and a major cause of morbidity and mortality due to its relatively poor prognosis. As part of ongoing studies addressing the molecular events underlying tumor progression in HNSCC, we have explored the nature of the proliferative pathways in which dysregulation may promote aberrant cell growth in this tumor type. The serine/threonine protein kinase Akt is a downstream target of phosphatidylinositol 3-kinase and a key regulator of normal and cancerous growth and cell fate decisions. Therefore, in this study, we have examined the status of activation of Akt in different stages of squamous cell carcinoma development in mice and in clinical samples from HNSCC patients. By immunohistochemical analysis, using a recently developed phosphorylation state-specific antibody, we demonstrated that Akt activation correlates closely with the progression of mouse skin squamous cell carcinoma. We also observed that activation of Akt is a frequent event in human HNSCC because active Akt can be detected in these tumors with a pattern of expression and localization correlating with the progression of the lesions. In line with these observations, Akt was constitutively activated in a large fraction of HNSCC-derived cell lines. We also provide evidence that the Akt signaling pathway may represent a biologically relevant target for a novel antineoplastic agent, UCN-01, which recently has been shown to be active in cellular and xenograft models for HNSCC at concentrations safely achievable in clinically relevant situations.

Background: Alterations in sex steroids during pregnancy are associated with the development and exacerbation of reactive lesions involving the gingiva. Currently, few experimental animal models similar to humans are available to examine regulatory pathways involving sex steroids and the periodontium. Methods: In the present study, we used the baboon as a novel experimental model for the study of the regulatory actions of estrogen on the periodontium during pregnancy. Pregnant baboons (N = 5) were administered the potent, highly specific aromatase inhibitor CGS 20267 (2 mg/day subcutaneously) daily on days 60 through 165 of gestation (term = 184). Untreated females (N = 10) and females (5) concomitantly administered aromatase inhibitor and estradiol benzoate (2.0 mg/day each subcutaneously) served as controls. Gingival biopsies were taken between days 145 and 165 of gestation. Results: Administration of CGS 20267 in all females suppressed maternal serum concentrations of estradiol by 95% and induced the development of an exuberant papillomatous enlargement of the gingiva by gestational day 110, with the most prominent development involving the labial aspects of the anterior sextants. None of the untreated pregnant controls or females concomitantly administered aromatase inhibitor and estradiol benzoate developed gingival overgrowth. Thus, estradiol alone prevented the onset of gingival overgrowth induced by estrogen suppression. In all baboons, discontinuation of the aromatase inhibitor at time of cesarean section resulted in spontaneous regression and resolution of the papillomatous hyperplasia within 4 to 6 weeks. Clinically, the gingival papillary overgrowth was erythematous and edematous, with a propensity toward spontaneous subgingival hemorrhage. Histologically, the biopsy specimens demonstrated hyperplasia of the epithelium typified by mild hyperkeratosis, acanthosis, and elongation and isolated anastamoses of rete ridges. Subjacent to the intact epithelium was a loose connective tissue stroma with isolated areas of inflammatory cell infiltrate. Special stains verified the presence of isolated bacterial biofilms; however, no evidence of fungal filaments was present. Histological features suggestive of viral infection were notably absent in the epithelium. No evidence of viral particles or capsids was identified using transmission electron microscopy. Reverse transcription polymerase chain reaction analysis, using a panel of degenerate primers, was negative for papilloma family viruses. Conclusions: These results are consistent with a significant role for estrogen during primate pregnancy in the regulation of cellular proliferation and differentiation within the gingiva. The baboon represents an important experimental model for studying the regulatory actions of estrogen on the periodontium during pregnancy. J Periodontol 2004;75:693-701.

Purpose We sought to evaluate the cell proliferation activity and immunohistochemical expression of proteins that promote or inhibit apoptosis in oral granular cell tumor (GCT). Study design Immunohistochemistry for Ki-67, a cell proliferation marker; Bcl-2, an anti-apoptotic protein; and caspase-3, a protein implicated in the execution of apoptosis, was performed on tissues from 12 patients with GCT of the tongue. Results Nuclear immunostaining for Ki-67 was observed only in isolated GCs (less than 2%). All patients exhibited cytoplasmic immunoreactivity for Bcl-2 in the majority of tumor cells. Cytoplasmic staining for caspase-3 was also present in more than 50% of GCs in all tumors. Conclusions GCT cells display low proliferation activity, a finding possibly related to their benign behavior. Caspase-3 expression suggests activation of the apoptotic cascade in the GCs, but persistence of the cells in the tissues could be attributed to the expression of Bcl-2 protein, a molecule that functions as a survival factor.

Background: The diffuse infiltrative lymphocytosis syndrome (DILS) in HIV patients is characterized by the persistence of CD8-circulating lymphocytes and lymphocytic infiltration, predominantly in salivary glands. Methods: We examined seven HIV-positive patients with bilateral parotid enlargement and sicca symptoms. Minor labial salivary gland biopsies were performed in all patients and submitted for histopathological analysis and immunohistochemistry for CD4, CD8, cytomegalovirus (CMV), LMP–EBV protein, and HIV p-24 protein. Results: In all cases, lymphocytic infiltration of the minor salivary glands, mainly periductal, was found. Acinar atrophy, ductal ectasia, and mild to moderate fibrosis were also observed. We noticed strong immunohistochemical reaction for LMP–EBV and p-24 proteins in ductal cells in all cases, while staining for CMV was consistently negative. The lymphocytes were positive for CD8, but consistently negative for CD4. Conclusions: A role of Epstein–Barr virus (EBV) and HIV, but not CMV, in the pathogenesis of DILS, is suggested by our immunohistochemical findings.

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.

Vitamin D-dependent rickets type I (VDDRI) represents an autosomal recessive hereditary defect in vitamin D metabolism. Patients with VDDRI have mutations of chromosome 12 that affect the gene for the enzyme 1-alpha-hydroxylase, resulting in decreased levels of 1,25(OH)(2) vitamin D. Clinical features include growth failure, hypotonia, weakness, rachitic rosary, convulsions, tetany, open fontanels, and pathologic fractures. The oral and dental manifestations of VDDRI have not been described. Here we present the case of a 10-year-old girl affected by VDDRI, as established by the combination of clinical and radiographic findings, family history, and laboratory values. Dental examination revealed markedly hypoplastic, yellowish-to-brownish enamel in all permanent teeth, malocclusion, and chronic periodontal disease. Large quadrangular pulp chambers and short roots were evident in dental radiographs. Light microscopic and ultrastructural examination showed abnormalities of dental hard tissues, affecting both enamel and dentin. The differential diagnosis and treatment of VDDRI are discussed.

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. Peptídeos nascentes de pró-colágeno e outras proteínas são transportadas por intermédio da membrana do retículo endoplasmático por um canal de transporte de proteínas chamado de tanslocon. Sec61alfa, uma proteína transmembrânica do translocon, tem sido apontada como essencial para translocação de cadeias nascentes de polipeptídeos para as cisternas do retículo endoplasmático. Entretanto, não se sabe se Sec61alfa é constitutivamente expressa em células de teratocarcinoma produtoras de colágeno. Além disso, a expressão e a utilização de Sec61alfa podem ser dependentes do estágio de diferenciação celular. Células progenitoras pluripotentes obtidas de muitas linhagens de células de teratocarcinoma, incluindo, por exemplo, células F9 e P19, são capazes de diferenciação em resposta a baixas concentrações de ácido retinóico. Essa diferenciação de células tumorais causa perda de sua carcinogenicidade. Para este estudo, células de teratocarcinoma F9 e P19 de camundongos foram cultivadas em meios de cultura tratados com ou sem ácido retinóico. A expressão de Sec61alfa foi determinada pelo método de "reverse transcriptase-polymerase chain reaction" (RT-PCR). Em condições não tratadas, células F9 expressaram quantidades não detectáveis de Sec61alfa. De forma similar, experimentos de RT-PCR demonstraram que a expressão gênica de Sec61alfa é estimulada nas células F9 após tratamento com ácido retinóico por 72 horas. Não foram detectadas diferenças na expressão de Sec61alfa nas células P19 após tratamento com ácido retinóico. Esses dados demonstram que a expressão de Sec61alfa é aumentada em células de teratocarcinoma F9 após diferenciação com ácido retinóico.

The aim of this study was to evaluate the histopathologic features and the expression of angiogenesis-related markers in primary tumors and metastatic lymph nodes of oral squamous cell carcinomas (SCCs) with multiple lymph node involvement in comparison with oral SCCs without nodal metastasis. The protein levels of the angiogenesis inhibitor endostatin, as well as those of the related molecules collagen XVIII, collagen-binding protein (CBP) 2/heat shock protein (HSP) 47, and cathepsin L, were evaluated by immunohistochemical analysis. Compared with nonmetastatic cases, primary tumors of the metastatic group exhibited significantly decreased protein levels of endostatin and its precursor collagen XVIII. Comparison between primary tumors and positive nodes of the metastatic cases revealed decreased expression of collagen XVIII and CBP2/HSP47 in metastases. Angiogenesis is essential for tumor growth and metastasis; accordingly, the observed differences in the immunohistochemical expression of angiogenesis-related proteins in oral SCC with multiple lymph node involvement may provide an explanation for the increased metastatic potential of these tumors.

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).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.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.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.

Activation of peroxisome proliferator-activated receptor gamma (PPARγ) has been linked to induction of differentiation, cell growth inhibition and apoptosis in several types of human cancer. However, the possible effects of PPARγ agonists on human oral squamous cell carcinoma have not yet been reported. In this study, treatment with 15-deoxy-Δ12,14-PGJ2 (15-PGJ2), a natural PPARγ 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 PPARγ 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 PPARγ ligands was also examined. Treatment of oral squamous cell carcinoma cells with 15-PGJ2 induced an initial reduction and eventual elimination of both phosphorylated and unphosphorylated Stat3 protein levels. In contrast, other PPARγ did not induce similar effects. Our results provide the first evidence of significant antineoplastic effects of 15-PGJ2 on human oral squamous cell carcinoma cells, which may be related to downmodulation of Stat3 and are at least partly mediated through PPARγ-independent events. British Journal of Cancer (2002) 87, 1396–1403. doi:10.1038/sj.bjc.6600618 www.bjcancer.com © 2002 Cancer Research UK

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. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:762-770, 2002

Leiomyosarcoma of the oral cavity is a very rare tumor that is associated with aggressive clinical behavior and low survival. In this paper, we report two new cases of leiomyosarcoma affecting the mandibular gingiva and mandible of a 35-year-old male and the mandible of a 51-year-old female. Given the difficulty in the histopathologic discrimination between benign and malignant smooth muscle tumors and the absence of reliable histologic parameters for prognostication of leiomyosarcomas, we evaluated the diagnostic and prognostic value of various immunohistochemical and molecular markers. By means of immunohistochemistry and quantitative real-time PCR analysis, we detected protein expression of PCNA, bcl-2, CDK4, p53 and MDM2 in both our cases and MDM2 amplification in our second case. The literature, pertinent to oral leiomyosarcoma and to molecular analysis of smooth muscle tumors, is reviewed.

Hereditary gingival fibromatosis (HGF) is a rare oral disease characterized by a slow and progressive enlargement of both the maxilla and mandible gingiva. Increased proliferation, elevated synthesis of extracellular matrix, particularly collagen, and reduced levels of matrix metalloproteinases seem to contribute to the pathogenesis of gingival overgrowth in HGF patients. Transforming growth factor-beta1 (TGF-beta1) is an important cytokine thought to play a major role in fibrotic disorders such as HGF due to its ability to stimulate the synthesis and reduce the degradation of extracellular matrix. In HGF fibroblasts, TGF-beta1 autocrine stimulation reduces expression and production of matrix metalloproteinases. However, the role of TGF-beta1 in fibroblast growth modulation has not been established in this disease.The aim of this study was to confirm the increased proliferation rate of HGF fibroblast cell lines and to explore a possible autocrine role of TGF-beta1 as a cell growth stimulator by blocking production of this endogenous cytokine using 2 well-established systems: antisense oligonucleotides and neutralizing antibodies.Four different cellular proliferation assays, bromodeoxyuridine labeling, argyrophilic nucleolar organizing region staining, proliferating cell nuclear antigen, and mitotic indexes, confirmed that fibroblasts from HGF proliferate significantly faster than those from normal gingiva. Antisense oligonucleotides reduced TGF-beta1 production as demonstrated by capture enzyme-linked immunosorbent assay, whereas TGF-beta1 expression levels were not significantly modified. Blocking TGF-beta1 synthesis with oligonucleotides or its activity with specific antibodies resulted in a decreased magnitude of HGF fibroblast proliferation.These results are consistent with the existence of an autocrine role of TGF-beta1 as a stimulator of HGF fibroblast proliferation.

Overexpression and amplification of several genes (MDM2, CDK4 and SAS) located on chromosome 12q13-15 have been noted to occur in various human sarcomas. As a result, two major growth regulation pathways may be inhibited. MDM2 may down regulate the p53-mediated growth control and CDK4 may affect pRB-mediated events. To determine the frequency of alterations in these genes and their correlation with clinicopathologic features, we analyzed the MDM2 and CDK4 protein levels by immunohistochemistry and assessed MDM2, CDK4 and SAS amplification by real-time PCR in nine osteosarcomas of the jaws. Positive staining for CDK4 and MDM2 was observed in eight cases (88.8%) and five cases (55.5%), respectively. Intense CDK4 staining was noted in four cases (two high grade, one intermediate grade and one low grade). Intense MDM2 staining was observed in the same four previous cases, as well as, one additional high-grade tumor. Individual DNA amplification for CDK4, MDM2 and SAS was observed in six cases for each gene. Co-amplification was observed in five cases that showed CDK4 and MDM2 concomitant amplification and four cases that displayed amplification for all of the genes. In addition, among the five cases that presented CDK4 and MDM2 amplification, strong overexpression of CDK4 and MDM2 was observed in three and in four cases, respectively (three high grade and one intermediate grade). These results suggest that 12q13-15 genes are involved in neoplastic disease and concurrent amplification and overexpression of these genes might help to define high-grade tumors.

Previously we have shown (Hebert et al. [1999] J. Cell Biochem. 73:248-258) that among many cell lines the CBP2 gene product, Hsp47, eludes its retention receptor, erd2P, resulting in the appearance of Hsp47 on the cell surface associated with the tetraspanin protein CD9. Since Hsp47 possesses a highly restricted binding cleft, random peptide display libraries were used to characterize peptides binding to Hsp47 and then to target this protein on carcinoma cell lines in vitro. Comparison of the clones obtained from panning revealed little specific homology based on sequence alone. To determine whether carcinoma cells expressing Hsp47 could selectively take up the selected bacteriophages, traditional immunofluorescence and confocal microscopy were employed. These studies revealed that phage-displaying Hsp47 binding peptides bound to cell lines expressing Hsp47 and that the peptides were rapidly taken up to a location coincident with Hsp47 staining. These observations were confirmed by cytometric analyses. These data indicate that CBP2 product may provide a molecular target for chemotherapy and/or imaging of malignancies.

Hereditary gingival fibromatosis (HGF) is characterized by an excess accumulation of extracellular matrix (ECM) resulting in a generalized and fibrotic enlargement of the gingiva. To investigate some of the regulatory features of this condition, gingival fibroblasts from normal gingiva (NG) and HGF were examined for the expression and production of matrix metalloproteinases (MMPs) and their inhibitors, tissue matrix metalloproteinases inhibitor (TIMPs). Our results, obtained from 2 different assays, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography, clearly demonstrated that the expression and production of MMP-1 and MMP-2 was significantly lower in fibroblasts from HGF than from NG. Interestingly, TIMP-1 and TIMP-2 expression from NG cells was shown to be slightly higher to those from HGF. Addition of antibodies against transforming growth factor-beta 1 (TGF-beta 1), which is produced in greater amounts by HGF fibroblasts, resulted in a slight increase in MMP-1 and a decrease in MMP-2 expression, whereas TIMP-1 and TIMP-2 expressions were unaffected. These patterns of expression and production suggest that enhanced TGF-beta 1 production reduce the proteolytic activities of HGF fibroblasts, which favor the accumulation of ECM.

Hsps expressed on the cell surface have been associated with tumor invasiveness and used as targets for molecular surveillance. The present study utilized four human oral squamous cell carcinoma cells lines, SCC-4, SCC-9, SCC-15, SCC-25, the murine epidermoid carcinoma cell line LL/2, and primary cultures of human gingival fibroblasts to assess the cell surface expression of colligin/Hsp47, a proposed marker for malignancy. Immunoprecipitation studies following protein crosslinking revealed that Hsp47 was associated with a number of membrane proteins including the tetraspanin CD9. Cytometric analyses were performed to determine the distribution of cell surface colligin/Hsp47 during the phases of the cell cycle. These studies showed that colligin/Hsp47 was not limited to any phase of the cell cycle in epidermoid carcinoma cells. Boyden chamber tumor invasion assays and colloidal gold migration assays utilizing a reconstituted basement membrane (Matrigel), collagen type I, and laminin-5 substrates revealed that cell lines expressing constitutive high levels of colligin/Hsp47 manifested the lowest invasion and migration indices. The incorporation of antibodies against Hsps into the migration and invasion assays, likewise, increased the invasion indices and the phagokinetic migration indices. These data indicate that colligin/Hsp47 is anchored to the cell membrane in a complex with CD9 where it moderates tumor cell invasion and motility possibly by acting as a serpin protein inhibitor or as a receptor for collagen.

HGF is a rare oral condition characterized by a slow, progressive enlargement of the gingiva, involving both the maxilla and mandible. HGF provides a model for the study of regulatory features of conditions characterized by connective tissue hyperplasia. In this study, the culture characteristics of gingival fibroblasts derived from patients of the same family with HGF (n = 4) were similar with regard to cell cycle analysis. Flow cytometric DNA content analysis revealed uniform DNA diploidy for fibroblasts cultured from NG and HGF. NG cells showed a low S-phase fraction (19.8%) and G2/M fraction (5.8%) and a relatively high G1 phase fraction (74%). In contrast, HGF cells from all members of the tested kindred, exhibited diploid cells with a higher S-phase (40.9%) and G2/M (10.1%) fraction and a relatively low G1 phase fraction (40.9%). Furthermore, we demonstrated that the expression and production of Hsp47 parallels the increased levels of collagen secretion observed in HGF. In addition, we show that Hsp47 and collagen are coordinately regulated following stress via a feedback mechanism mediated by N-terminal procollagen propeptides. Utilizing confocal microscopy and antibodies directed against GST-fusion proteins encompassing the pro alpha1(I) N-propeptide globular domain (NP1) (residues 23-108), it was apparent that this regulatory mechanism does not involve significant interaction with Hsp47's chaperoning of procollagen.

Hsp47 is a novel glycoprotein that binds specifically to procollagen and is retained in the ER by its COOH-terminus RDEL peptide sequence (Satoh, M. et al. Jol. Cell Biol. 1996; 133: 469-83). In this paper, we report that erd2P, the KDEL receptor, is distributed, coprecipitates with, and binds to Hsp47. Also, under stress conditions and lowering of pHi, the cytoplasmic epitope of erd2P is not recognized by erd2P antibodies unless the cells are pretreated with NEM. Coincident with the masking of the cytoplasmic epitope of erd2P, following lowering of pHi, Hsp47 is not retained but eludes its retention receptor to be expressed on the cell surface. Alkalization of the endosomal compartments by treatment with NH4Cl or chloroquine also results in the loss of Hsp47 to the cell surface, presumably by inhibiting the retrieval of trans-Golgi network proteins from the cell surface. The expression of Hsp47 on the cell surface under conditions of stress and alteration of pHi and pHe posture Hsp47 as a serpin family protein that may modulate cell migration during development and invasion and metastasis in cancer.

Collagen type IV is a genetically distinct secretory protein that constitutes a major component of the basement membranes. Despite the differences between collagen types I and IV, it appears that collagen IV alpha-chain translation/translocation through the ER should follow similar pathways to those established for procollagen I. Using a collagen IV-producing mouse teratocarcinoma cell line, we sought to determine if evolving nascent chains of collagen IV are associated with Hsp47 and other ER-resident molecular chaperones. We show that Hsp47, Grp78 and Grp94 appear to form heteromeric complexes associated with each other and with nascent chains of collagen IV. In addition, we investigated whether ATP depletion, a condition known to promote stable association of Grp78 and Grp94 proteins with their chaperone substrates, extended this response to include a chaperone independent of ATP, Hsp47. These studies reveal that ATP depletion increased the levels of newly synthesized collagen IV associated with Hsp47, Grp78, Grp94 and with each other. These results indicate that Hsp47, Grp78 and Grp94 exist as oligomers and form heterocomplexes during the maturation of newly synthesized collagen IV chains.

Objective. Heat shock proteins (Hsps), a highly conserved class of protective cellular proteins that are produced under various conditions of environmental challenge, have been implicated as the antigenic stimulus in autoimmune diseases. Because lichen planus (LP) appears to be an autoimmune or hyperimmune condition (mediated by T cells), Hsps may have a role in the pathogenesis of this disease. We believe that if keratinocyte Hsps are antigenic targets of a cellular immune response, upregulation of these proteins should be demonstrable in tissue sections. Study design. Immunohistochemistry was used to evaluate expression of several families of Hsps in oral lichen planus tissues. The number and distribution of γδ T cells, a subset of T lymphocytes with an immune surveillance function that may contribute to autoimmunity, were also evaluated. Monoclonal antibodies to Hsps 27, 60, 70, 90, γδ receptor, and CD3 (pan-T lymphocyte marker) were incubated with frozen sections of LP ( n =22) and normal oral mucosa ( n =17) followed by an avidin-biotin-peroxidase labeling method. Antibodies to bacterial Hsps (GroEL and DnaK) were used as negative controls, and antibody to constitutive eukaryotic Hsp (Hsc70) was used as a positive control. Results. In six cases of LP, basal keratinocytes stained intensely for Hsp27, whereas controls showed only slight staining. Otherwise LP and normal tissues showed comparable positive staining of upper level keratinocytes with anti-Hsp27. Subjective increases in antibody staining were noted for Hsp60 in LP, which was due in part to staining of infiltrating lymphocytes and in part to keratinocyte expression. Normal tissues showed weak basal cell antibody staining for Hsp60. Hsp70 staining was observed at a less intense level in LP than in controls. Except for more intense basement membrane staining with anti-Hsp90 antibody in gingiva and palate, no differences in the occurrence of this protein were found. Absolute numbers of γδ T cells were increased in LP when compared with those in control specimens ( n =10 vs n =1, respectively, per high-power field). However, γδ T cells represented less than 1% of the CD3+ lymphocytes. Conclusions. It was concluded that normal oral mucosa expresses Hsps 27, 60, 70, and 90 and contains few γδ T cells. Although the expression of Hsps was altered in LP, the differences demonstrated were slight and were therefore inconclusive. The Hsps expressed in LP could have contributed to the persistence or chronicity of the disease, or they could have simply reflected cellular injury.

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts, has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with 35[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro alpha 1(I)-N-propeptide at 25 micrograms/ml completely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86-90 of the pro alpha 1(I)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro alpha 1(I)-N-propeptide domain were confirmed by examination of polysome-associated pro alpha chains. The nascent pro alpha 1(I)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro alpha 1 (I)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23-108 (NP1), 23-151 (NP2), and 23-178 (NP3) within the pro alpha 1 (I)- N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro alpha 1(I)- and pro alpha 2(I)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro alpha 2(I)-chains and hsp47, whereas there was strong interaction between pro alpha 1(I)-chains and nascent hsp47. SP1.D8 could not precipitate pro alpha 1(I)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a "chaperone" role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the pro alpha 1 (I) chain, and the pro alpha 1(I) amino-propeptide region in particular.

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated α1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5ʹ-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.

Hsp47, Grp78, and Grp94 have been implicated with procollagen maturation events. In particular, Hsp47 has been shown to nascent procollagen alpha 1(I) chains in the course of synthesis and/or translocation into the endoplasmic reticulum (ER). Although, Hsp47 binding to gelatin and collagen has previously been suggested to be independent of ATP. Grp78 and Grp94 are known to dissociate from its substrates by an ATP-dependent release mechanism. The early association of Hsp47 with procollagen and its relatively late release suggested that other chaperones, Grp78 and Grp94, interact successively or concurrently with Hsp47. Herein, we examined how these events occur in cells metabolically stressed by depletion of ATP. In cells depleted of ATP, the release of Hsp47, Grp78, and Grp94 from maturing procollagen is delayed. Thus, in cells experiencing metabolic stress, newly synthesized procollagen unable to properly fold became stably bound to a complex of molecular chaperones. In that Hsp47, Grp78, and Grp94 could be recovered with nascent procollagen and as oligomers in ATP depleted cells suggests that these chaperones function in a series of coupled or successive reactions.

Hsp47, an endoplasmic reticulum resident protein, has gelatin-binding and procollagen-binding properties and has been hypothesized to function as a molecular chaperone in regulating procollagen folding and/or assembly. In this report, we further investigate the interaction of Hsp47 with polysome-associated alpha 1(I) procollagen chains following antisense treatment of 3T6 cells. For these studies, we employed phosphorothioate oligodeoxynucleotides directed to the first five codons of Hsp47 that straddle the predicted translation initiation site of mouse Hsp47. Cells depleted of Hsp47 in this manner were observed to produce diminished amounts of fully elongated nascent alpha 1(I) procollagen while accumulating shorter procollagen peptides associated with peptidyl-tRNA. Pulse-labeling of cells with [35S]methionine followed by treatment with puromycin and immunoprecipitation with anti-Hsp47 and anti-procollagen antibodies revealed that Hsp47 is associated with alpha 1(I) procollagen at a very early point during translocation of the nascent procollagen chains. Although Hsp47 appears to possess properties similar to grp78/BiP, Hsp47 binding early during translocation favors a more specialized specific function relative to chain selection or completion of stable folding in type I procollagen.

Background. Alterations in the tumor suppressor gene p53 are the most frequently detected genetic abnormalities in human cancers. Inactivated tumor suppressor genes, including p53, often are suggested by loss of heterozygosity (LOH) studies. p53 gene inactivation has been reported in esophageal cancers. Because the etiologic factors for esophageal and intraoral carcinomas often are the same, corresponding molecular events may occur in oral squamous cell carcinoma (SCC) development. Methods. The authors investigated LOH of the p53 gene in DNA from 27 primary oral cancers using a polymerase chain reaction (PCR)-based restriction fragment length polymorphism assay. DNA from fixed specimens of SCC and normal tissues was isolated and amplified at two p53 gene polymorphic restriction sites. Results. In heterozygous individuals, 10 of 14 (71%) intraoral SCC demonstrated loss of p53 heterozygosity at one polymorphic restriction site. Two of five carcinomas showed LOH at a second site. Conclusions. These results suggest that inactivation of p53 is involved in the development or progression of SCC of the oral cavity.

To determine whether the proposed molecular chaperone Hsp47 is associated with the production of heterotrimeric procollagen, the distribution of anti-Hsp47 and anti-collagen antibodies were examined in developing murine femurs and molars of 22-23-day CD-1 mice. In addition, the expression of Hsp47, and collagen mRNAs were assessed by in situ hybridization using oligonucleotide probes. These studies revealed that Hsp47 was developmentally expressed and produced in regions that are coincident with type I collagen. Hsp47 was not localized in cartilaginous zones of developing femurs or in the regions of developing molars producing type III collagen. These results support the hypothesis that Hsp47 is necessary for the assurance of type I procollagen and is not expressed with other homotrimeric procollagen molecules.