Gain of 11q/cyclin D1 overexpression is an essential early step in skin cancer development and causes abnormal tissue organization and differentiation.

Non-melanoma skin cancers, in particular keratoacanthomas (KAs) and squamous cell carcinomas (SCCs), have become highly frequent tumor types especially in immune-suppressed transplant patients. Nevertheless, little is known about essential genetic changes. As a paradigm of 'early' changes, that is, changes still compatible with tumor regression, we studied KAs by comparative genomic hybridization and show that gain of chromosome 11q is not only one of the most frequent aberration (8/18), but in four tumors also the only aberration. Furthermore, 11q gain correlated with amplification of the cyclin D1 locus (10/14), as determined by fluorescence in situ hybridization, and overexpression of cyclin D1 protein (25/31), as detected by immunohistochemistry. For unraveling the functional consequence, we overexpressed cyclin D1 in HaCaT skin keratinocytes. These cells only gained little growth advantage in conventional and in organotypic co-cultures. However, although the control vector-transfected cells formed a well-stratified and orderly differentiated epidermis-like epithelium, they showed deregulation of tissue architecture with an altered localization of proliferation and impaired differentiation. The most severe phenotype was seen in a clone that additionally upregulated cdk4 and p21. These cells lacked terminal differentiation, exhibited a more autonomous growth in vitro and in vivo and even formed tumors in two injection sites with a growth pattern resembling that of human KAs. Thus, our results identify 11q13 gain/cyclin D1 overexpression as an important step in KA formation and point to a function that exceeds its known role in proliferation by disrupting tissue organization and thereby allowing abnormal growth.

Phenotypic comparison of periodontal ligament cells in vivo and in vitro.

The mammalian periodontal ligament contains heterogeneous populations of connective tissue cells, the precise function of which is poorly understood. Despite close proximity to bone and the application of high amplitude physical forces, cells in the periodontal ligament (PL) are capable of expressing regulatory factors that maintain PL width during adult life. The study of PL homeostasis and PL cell differentiation requires culture and phenotypic methods for precise characterization of PL cell populations, in particular those cells with an inherently osteogenic program. Currently it is unknown if cells cultured from the PL are phenotypically similar to the parental cells that are present in the tissues. We have compared the phenotype of cells in vivo with cells derived from the PL and expanded in vitro to assess the general validity of in vitro models for the study of phenotypic regulation in vivo. Rat PL cells were isolated by either scraping the root of the extracted first mandibular molars (Group A), or by scraping the alveolar socket following extraction of first mandibular molars (Group B), or by obtaining a mixture of cells after disaggregating a block of tissue consisting of first mandibular molar, PL and the surrounding alveolar bone (Group C). Cultured cells at confluence were fixed and immunostained for alpha-smooth muscle actin (alpha-SMA), osteopontin (OPN), alkaline phosphatase (AP), or bone sialoprotein (BSP). For in vivo assessments, frontal sections of rat first mandibular molar were immunostained for alpha-SMA, OPN, AP and BSP. We examined osteogenic differentiation of cultured PL cell cultures by bone nodule-forming assays. In vivo and at all examined sites, > 68% of PL cells were immunostained for AP; approximately 50% and approximately 51% for OPN and alpha-SMA (p = 0.3), respectively, while only approximately 8% were positively stained for BSP (p < 0.01). Analysis of cultured PL cells in Groups A, B and C showed 54%, 53%, and 56% positive staining for alpha-SMA respectively; 51%, 56%, 54% for OPN; 66%, 70%, 69% for AP and 2.2%, 1.4% and 2.8% for BSP. The mean percentage of PL cells in situ stained for the different markers was similar to that of cultured PL cells (Group A approximately Group B approximately Group C in situ for p > 0.2) except for BSP which was 3 to 4 fold higher in vitro (p < 0.01). PL cell cultures treated with dexamethasone showed mineralized tissue formation for all groups (A, B, C), but no mineralized tissue formation was detected in the absence of dexamethasone. As PL cells express quantitatively similar phenotypes in vitro and in vivo, we conclude that the in vitro models used here for assessment of PL cell differentiation appear to be appropriate and are independent of the cell sampling method. Further, dexamethasone-dependent progenitors are present both on the root and bone-related sides of the PL.

Oral bowenoid lesions: differential diagnosis and pathogenetic insights.

OBJECTIVE: To determine if oral lesions exhibiting bowenoid features reflect the diverse microscopic appearance and biologic behaviour of Bowen's disease and bowenoid papulosis of the skin and genitalia. STUDY DESIGN: Seven cases of oral bowenoid lesions (6 with follow-up data) were assessed for differences in histologic features, human papillomavirus (HPV) viral status, and selected immunohistochemically detectable cell cycling proteins (p53, WAF-1, Cyclin D1, Bcl-2) and were correlated with available follow-up data. RESULTS: Two histologic subsets were identified. One, which was believed to correspond to Bowen's disease, exhibited large numbers of transepithelial apoptotic bodies, dyskeratotic cells and mitoses (bowenoid elements), poor differentiation of background epithelial cells, and consistent HPV-16/18 positivity. The other, believed to correspond to bowenoid papulosis, exhibited few bowenoid elements, good background differentiation, and inconsistent HPV-16/18 positivity. One of the aggressive cases exhibited repeated recurrences despite apparent total clinical excision, whereas none of the other group recurred. CONCLUSION: Although a small number of cases are in this study, results suggest that oral bowenoid lesions may exhibit histopathologic and behavioral variations ranging from oral Bowen's disease to oral bowenoid papulosis. Studies on more cases are needed to confirm this initial impression.

Herpesvirus-induced diseases: oral manifestations and current treatment options.

The dentist is often the first health professional to be contracted by patients who develop acute orofacial symptoms of viral conditions such as shingles (varicella zoster) or herpetic gingivostomatitis. The diagnosis, treatment, and management of virally induced oral diseases is a challenge inasmuch as their presentation is atypical and may be complicated by immunosuppression. However, an increasing body of knowledge regarding the manifestations of viral infections in immunocompromised patients and the advances achieved in antiviral drug therapy during the past several years should make the task less daunting for the dentist. In this paper, the natural history, typical and atypical oral manifestations, diagnosis, current treatment options, and advances in the prevention of common herpesvirus-induced diseases are reviewed, with particular attention to primary and recurrent varicella zoster virus and herpes simplex type 1 infections.

Detection of Helicobacter pylori in oral aphthous ulcers.

A causative role for Helicobacter pylori (H. pylori) in the pathogenesis of oral mucosal ulcerations has been suggested previously. We have adopted the polymerase chain reaction (PCR) as a rapid and sensitive means to detect H. pylori in swabs of recurrent oral aphthous ulcers and in samples of other oral sites. Of the oral aphthous ulcer samples, 32 (71.8%) were found to be positive, while the saliva and plaque samples (most of them taken from the patients with aphthous ulcers) were consistently negative for H. pylori DNA, as detected by the PCR assay. Only two of the swab samples from the tongue (collected at the time of concurrent, H. pylori-positive oral aphthous ulcers) were found to be positive. The data suggest that H. pylori may be associated frequently with recurrent oral aphthous ulcers, and are consistent with previous studies indicating that saliva and plaque are not likely sources of contamination with this microorganism. There was no apparent correlation with HIV status (infection with human immunodeficiency virus). The possible pathogenic significance of Helicobacter pylori in oral ulcerations is discussed.

Human papilloma virus type 16 E6 gene expression in oral exophytic epithelial lesions as detected by in situ rtPCR.

OBJECTIVE: We adopted an in situ reverse transcriptase polymerase chain reaction method of detecting and determining the frequency of early (E6) gene expression of human papilloma virus type 16 at the individual cell level in a sample of oral exophytic lesions with various degrees of epithelial hyperplasia and dysplasia in immunosuppressed and immunocompetent patients. STUDY DESIGN: The significance of differences between the study groups was determined by Mantel-Haenszel chi-square analysis and calculation of odds ratios, accounting for immunosuppression and degree of dysplasia, respectively. RESULTS: Grouped together, the lesions of dysplasia (mild to severe) and squamous cell carcinoma were found to be 16 times more likely to express human papilloma virus E6 mRNA than the benign lesions (P = .0013); in the lesions of immunosuppressed patients, human papilloma virus 16 E6 was roughly 10 times more likely to be expressed than in those of the immunocompetent patients (P = .0008, accounting for dysplasia). CONCLUSIONS: Our data indicate that human papilloma virus 16 E6 gene expression, and perhaps integration of the virus in the host genome, might play a role in the development of oral neoplasia in association with immunosuppression.

Differentiation and the cytomorphology of salivary gland tumors with specific reference to oncocytic metaplasia.

OBJECTIVE: The different cell types and many growth patterns found in salivary gland tumors provide ample reason for the diagnostic problems caused by these tumors. To improve criteria for differential diagnosis, the potential range of cytologic features possible in salivary gland tumor cells must be better appreciated. STUDY DESIGN: From our respective pathology archives, normal salivary tissue and salivary gland tumours--other than Warthin's tumor and oncocytoma--with oncocytic differentiation were identified and studied by means of light and electron microscopy. RESULTS: In this article, we cite a number of different salivary gland tumors, including basal cell adenoma, pleomorphic adenoma, myoepithelioma, polymorphous low-grade adenocarcinoma, and mucoepidermoid carcinoma, showing varying degrees of oncocytic differentiation. CONCLUSIONS: Variable cellular differentiation is probably the basis for foci of tumor cells unexpected for a particular salivary gland neoplasm, further compounding differential diagnosis. Illustration of oncocytic differentiation serves 2 purposes. First, it can alert pathologists to this potential in otherwise typical salivary gland tumors; an awareness of this and other possible variations in cellular differential patterns can help prevent misdiagnosis. Second, these particular tumors illustrate the role of the cellular differentiation that is responsible for the range of histologic features within any one subtype of salivary gland tumors.

Demonstration of c-erbB-2 oncogene overexpression in salivary gland neoplasms by in situ hybridization.

The level of c-erbB-2 cellular mRNA in 18 salivary gland tumours and in 7 normal salivary glands was determined by in situ hybridization using [35S] labelled RNA probes. Computer assisted quantitation of the autoradiographic signal indicated a significantly higher c-erbB-2 expression in the tumour group (22.64 grains per cell +/- 3.79; 95% CI) as compared to the non-neoplastic salivary gland tissue (4.11 +/- 0.90; 95% CI). The c-erbB-2 expression as measured by grain counts per cell for the pleomorphic adenomas (16.29 +/- 1.87; 95% CI), mucoepidermoid carcinomas (31.52 +/- 0.08; 95% CI) and the acinic cell carcinomas (44.24 +/- 17.11; 95% CI) were significantly greater than the expression for the normal group. The acinic cell carcinomas exhibited the greatest level of expression. As observed at the individual cell level, the autoradiographic signal was distributed uniformly in the neoplastic tissues, regardless of the cell type. This study confirms the hypothesis that the c-erbB-2 oncogene is overexpressed at the mRNA level in salivary gland tumours.

Analysis of c-erbB-2 amplification in salivary gland tumours by differential polymerase chain reaction.

DNA samples extracted from 22 normal salivary glands, 38 salivary pleomorphic adenomas and 20 other salivary gland neoplasms were screened for amplification of the c-erbB-2 oncogene by a differential polymerase chain reaction (PCR). The samples were PCR amplified with primers specific for the c-erbB-2 oncogene and for a reference gene (interferon-gamma). A breast carcinoma cell line SKBR-3 known to contain c-erbB-2 amplification was used as positive control. Following gel electrophoresis, the intensity of the amplified DNA bands was determined by laser densitometry and the level of amplification of the c-erbB-2 oncogene was assessed from the intensity of the c-erbB-2 specific band relative to that of the interferon-gamma band. Of all the tumours detected, only the two poorly differentiated adenocarcinomas, two of the pleomorphic adenomas and one of the Warthin's tumours showed gene amplification at levels comparable to the breast carcinoma cell line. None of the normal salivary gland tissues was found to have amplification. Within the group of pleomorphic adenomas the average level of amplification was not significantly different from that observed in the normal salivary gland, or in total genomic DNA from unrelated tissue (P < or = 0.001, determined by a general linear model of statistical analysis). These results indicate that amplification of the c-erbB-2 oncogene is infrequent in salivary neoplasia. Thus, gene amplification alone cannot account for the high prevalence of c-erbB-2 overexpression demonstrated previously in salivary gland tumours. When present, c-erbB-2 amplification may be associated with a more aggressive behaviour.

c-fos oncogene underexpression in salivary gland tumors as measured by in situ hybridization.

Tissue from 35 salivary gland tumors and 14 normal salivary glands was analyzed by in situ hybridization and computer-assisted morphometry for the expression of the c-fos oncogene. The normal salivary gland tissues were found to express c-fos focally, mainly in the acinar secretory cells. The majority of the cells in the normal tissues showed a high level of expression (47.74 +/- 5.31% of cells had 46 to 60 grains per cell and another 45.79 +/- 2.18% showed > 60 grains per cell). All the tumors examined exhibited a relatively low, uniform distribution of c-fos expression. For example, in the poorly differentiated adenocarcinomas, 96.83 +/- 04% of the cells were found to have < 15 grains per cell. A general linear model for multivariate analysis showed a significant difference between the various tumor types and the normal salivary gland tissues (P = 0.0001). These data support the hypothesis that salivary gland tumors belong to a group of epithelial neoplasias in which the loss of cellular differentiation is linked with underexpression of the c-fos oncogene.

c-fos oncogene expression in dexamethasone stimulated osteogenic cells in chick embryo periosteal cultures.

Although the complex effects of glucocorticoids on bone cells have been studied extensively in vitro, little is known about the molecular mechanisms of glucocorticoid responses in osteogenic cells. As c-fos and its protein product are believed to play a key role in intracellular signal transduction, and since their role in regulation of bone formation is well-recognized, we studied the effect of the glucocorticoid analogue dexamethasone (DEX) on the expression of c-fos oncogene in the chick periosteal osteogenesis (CPO) model. C-fos mRNA expression was determined by in situ hybridization at various time points after 10(-7) M DEX treatment. Prior to DEX treatment, the cultures had been synchronized with 2 mM thymidine. The mean area of positively hybridized cells in experimental (DEX-treated) and control (DEX-free) cultures was quantitated by computer assisted morphometry. In DEX-treated cultures c-fos mRNA could be detected transiently and mainly in the osteogenic layer at 30, 45 and 60 min after treatment whereas no c-fos expression could be detected above background level in the control groups. Differences between experimental and control groups were significant (P less than 0.01) as determined by a general linear model (GLM) analysis of variance. These data indicate that in the CPO culture system, DEX (10(-7) M) induces c-fos expression. The findings are compatible with the hypothesis which states that glucocorticoid-induced phenotypic changes in osteogenic cells may be mediated by c-fos.

Inducibility of neoplastic transformation by Fujinami sarcoma virus in an in vitro chick embryo model for osteosarcoma: (i) effect of differentiation and (ii) investigation for in vivo growth potential in athymic mice.

We have described previously a novel in vitro model for the study of osteosarcoma. In this system, chick periosteal explants (CEP) transformed by the P140gag-fps oncoprotein of Fujinami avian sarcoma virus (FSV) exhibit biochemical and histological manifestations characteristic of osteosarcoma. In the present study, a hypothesis suggesting that more differentiated bone cells may resist FSV-induced oncogene changes was tested. In one set of experiments, CEP cultures were pretreated with a high dose of dexamethasone (10(-7) M), a bone cell differentiating agent, prior to FSV infection. In another experiment, CEP explants were allowed to grow and thus differentiate for various lengths of time in culture prior to infection with FSV. Another goal of this study was to show that FSV-transformed cultures were tumorigenic in nude mice. In experiments focusing on differentiation and FSV-transformation, it was found that groups that had been infected at stages where osteogenic differentiation had been induced or allowed to occur, exhibited significantly decreased values for biochemical parameters associated with osteosarcomatous transformation. Specifically, these parameters were alkaline and acid phosphatase activity, protein content, [3H]thymidine incorporation, mineral profile, and acidification of culture media. Furthermore, osteosarcomatous histopathological features were more prominent in cultures subjected to FSV infection prior to differentiation. These findings indicate that differentiated osteogenic cells are less susceptible to oncogene-mediated transformation than their progenitors. The tumorigenic potential of some CEP cultures transformed in vitro with FSV was examined by transplantation into athymic mice. FSV-transformed CEP cultures xenografted subcutaneously exhibited tumor formation, whereas xenografts of uninfected cultures did not grow or were completely resorbed. This demonstrates that FSV-transformed cultures are tumorigenic, and confirms that this model system is useful for the investigation of the mechanisms governing the development of osteosarcoma in vitro.

EBV and HSV infections in a patient who had undergone bone marrow transplantation: oral manifestations and diagnosis by in situ nucleic acid hybridization.

The course of infections with herpes simplex virus and Epstein-Barr virus in an immunosuppressed patient who had undergone bone marrow transplantation and had tested seronegative for human immunodeficiency virus is described. The clinical oral manifestations were unusual, as they included hairy leukoplakia-like lesions and extensive mucosal ulceration. Histologic examination disclosed unique features consisting of both lichenoid and viral cytopathic changes. The association of the lesions with both Epstein-Barr virus and herpes simplex virus was confirmed by in situ hybridization histochemistry. The importance of recognition of the symptoms, specific diagnosis by DNA hybridization, and implications for antiviral prophylaxis and therapy are emphasized.

The regulatory effect of phosphates on bone metabolism in vitro.

One of the most important indicators in vitro of the bone-cell phenotype is the synthesis of mineralized bone-like tissue. This has been achieved by supplementing isolated bone-cell and tissue cultures with organic phosphates, in particular, beta-glycerophosphate. To analyze the effects of beta-glycerophosphate on bone-cell metabolism and osteogenesis in vitro, both biochemical analyses and computer-assisted morphometry were used. Simultaneous autoradiographic and histochemical analyses of proliferating and alkaline phosphatase-positive cells were used to measure osteogenic events at the cellular level. Morphometric data showed that beta-glycerophosphate-treated cultures mineralized, but exhibited significantly less bone matrix (P less than 0.05) than non-mineralizing controls. Cultures treated with inorganic phosphate failed to mineralize. Cellular proliferation was unaffected by beta-glycerophosphate; however, there was a decrease in the amount of 3H-thymidine incorporation into the DNA of beta-glycerophosphate-treated cells as detected by autoradiography. The percentage of alkaline phosphatase-positive cells was identical in beta-glycerophosphate-treated or control cultures. In agreement with previous biochemical results, there was a decrease in the amount of alkaline phosphatase enzyme activity per cell. The kinetics of alkaline phosphatase enzymes were measured on individual cells by microdensitometry. beta-Glycerophosphate-treated cultures exhibited more rapid reaction rates than control cultures (p less than 0.05). Taken together, the results suggest that beta-glycerophosphate has global effects on bone-cell metabolism in vitro including its importance in mineralization.

Site-specific regulation of osteogenesis: maintenance of discrete levels of phenotypic expression in vitro.

Intrinsic differences in bone formation rate, cell numbers, and the percentages of cells expressing alkaline phosphatase activity were studied in explants of chick calvaria periosteum cultured for 4 days and 6 days. Proliferation, differentiation, and bone production were examined in radioautographs of plastic sections and by using whole-culture biochemical assays of protein and alkaline phosphatase. Ectocranial explants at both 4 days and 6 days exhibited more alkaline phosphatase-positive cells and significantly more bone formation than endocranial cultures. There were no detectable differences in cell numbers or 3H-thymidine labeling indices. The volume of bone synthesized per osteoblast was significantly higher in the ectocranial group. Examination of bone stripped of periostea and then cultured for 4 days revealed that large areas of bone were covered by osteoblasts, indicating that the periosteal explant cultures were composed almost exclusively of osteoprogenitor cells and fibroblasts. The data suggest that the level of expression of predetermined osteogenic phenotypes can be maintained in vitro for 6 days following explantation and that variations in the rate of osteogenesis are programmed into progenitor cells prior to their differentiation into osteoblasts.

Dexamethasone effects on induction of neoplastic transformation by Fujinami sarcoma virus in an in vitro chick embryo periosteal model for osteosarcoma.

Recently we have developed a model in vitro system for the study of factors regulating the histogenesis of osteosarcoma. In this system, Fujinami sarcoma virus (FSV) induces osteosarcomatous changes such as increased cell proliferation and altered patterns of bone and nonmineralized matrix (osteoid) formation. Such changes can be quantitated at the individual cell level, by computer-assisted morphometry. Here we report on the effects of dexamethasone (DEX) on FSV-induced neoplastic transformation and osteogenesis in chick embryonic periosteum cultures, as reflected by a series of histopathological parameters. Most significantly, it was found that compared to 10(-9) M DEX treated cultures, in 10(-7) M DEX pretreated cultures, the bone/osteoid ratio was increased due to a relative increase in the area of bone and a decrease in the area of osteoid. The number of [3H]thymidine-labeled cells decreased significantly, while the proportion of alkaline phosphatase positive cells increased. Double-label immunohistochemistry (with anti-P140gag-fps) and histochemistry for alkaline phosphatase activity was performed, to demonstrate production of the oncogene-encoded protein, and osteoblastic differentiation, respectively. In an in vitro transformation assay single cells derived from 10(-9) M DEX treated cultures formed a significantly higher number of colonies than those obtained from 10(-7) M DEX pretreated cultures. Taken together, the data indicate that in the chick embryonic periosteum culture system, pretreatment with 10(-7) M DEX inhibits the ability of FSV to induce neoplastic transformation. This effect is probably the result of DEX-induced cell differentiation, prior to infection with FSV.

Neoplastic transformation of osteogenic cells: quantitative morphometric analysis of an in vitro model for osteosarcoma.

Previously we have reported the development of a model in vitro system for the study of osteosarcoma. In this system, when chick periosteal explants are infected with Fujinami sarcoma virus (FSV), osteosarcoma-like tissue is formed. In the present study, a series of histopathologic parameters of neoplastic transformation and osteogenesis were quantitated, at a single cell level, by computer-assisted morphometry. Most significantly, it was found that compared to uninfected (control) cultures, in the FSV-infected (experimental) cultures, the bone to osteoid ratio per unit area was decreased due to a relative decrease in the area of bone and an increase in the area of osteoid. The cellularity of the FSV-infected tissues was significantly increased due to an increase in the number of unlabeled and [3H]thymidine-labeled cells, while the proportion of alkaline phosphatase (AP) positive cells decreased. Double-label immunohistochemistry (with anti-P140gag-fps) and histochemistry for AP activity was performed, to demonstrate production of the oncogene-encoded protein, and osteoblastic differentiation respectively. In an in vitro transformation assay, single cells derived from control, uninfected cultures did not grow, while those derived from FSV-infected cultures formed colonies in semisolid medium. Some of these colonies demonstrated AP staining. Taken together these data show that in this in vitro system (i) neoplastic transformation of osteogenic cells does occur, (ii) changes in osteoid and bone production are related to neoplastic transformation, and (iii) osteosarcoma-like changes can be quantitated at the individual cell level.

Two cases of oral pigmentation associated with quinidine therapy.

Melanin pigmentation of the hard palate is described in two patients receiving long-term quinidine therapy for cardiac arrhythmia. The importance of quinidine-induced oral pigmentation in the differential diagnosis of oral pigmentary disturbances is discussed.

In vitro transformation of osteoblasts: putative formation of osteosarcoma in vitro.

The study of bone cancer has been difficult in part due to a lack of appropriate in vitro osteosarcoma model systems. The development of such systems is essential if a clearer understanding of the biology of and mechanisms behind the formation and progression of bone cancers is to be obtained. We report here the development of an in vitro model system which demonstrates important characteristics generally associated with osteosarcoma. The chick periosteal osteogenesis model was infected with the Fujinami Sarcoma Virus (FSV) containing the v-fps oncogene which encodes for a P140gag-fps protein-tyrosine kinase. Under the appropriate conditions FSV infected cultures developed bone and cartilaginous tissues which showed histopathological findings consistent with osteosarcoma. Biochemical data indicating massive increases in alkaline phosphatase activity, protein content, 3H-Thymidine incorporation as well as expression of active P140gag-fps confirm that transformation has occurred in FSV infected cultures. This novel in vitro model system should prove most useful in the study of bone cancer.