Semiquantitative morphological analysis of stromal cells in the irradiated and recovering rat thymus.

To understand the roles of thymic stromal cells in T-lymphocyte development, we semiquantitatively analysed rat thymi recovering from irradiation (6 Gy), using a transmission electron microscope. The most striking findings were that the percentage of subcapsular epithelial cells significantly increased in the cortex on day 3 after irradiation compared with the control; the percentage of intermediate epithelial cells significantly increased in the cortex on days 3 and 5 after irradiation and in the medulla on days 5 and 7 compared with the control; the interdigitating cells disappeared from the medulla by day 7 after irradiation and reappeared on day 9. The present data thus reveal that during recovery after irradiation (6 Gy), marked changes occur in the relative proportions of different epithelial cell subtypes in the cortex and medulla of the rat thymus. In addition, the percentages of macrophages and interdigitating cells also changed during the recovery. These changes, which may be associated with the abrupt proliferation of thymocytes after irradiation, should shed light on the significance of stromal cells in the T cell development.

The relationship of genetic aberrations detected by comparative genomic hybridization to DNA ploidy and tumor size in human oral squamous cell carcinomas.

We have examined genetic alterations in 11 surgically removed oral squamous cell carcinomas (OSCCs) using comparative genomic hybridization (CGH) and laser scanning cytometry (LSC), which allow quantitative analysis of chromosomal abnormalities. CGH analysis revealed gains and/or losses of DNA sequence copy number in all tumors. Gains in DNA sequence copy number were detected frequently for chromosome arms 3q25-28 (6/11), 5p (6/11) and 8q (5/11), and losses in chromosome arms 18q (4/11), 19q (4/11), 17p (3/11), and 19p (3/11). Amplification of 5p was observed in two tumors. LSC detected DNA aneuploidy with DNA indices ranging from 1.30 to 1.82 in 6 of 11 tumors. The number of chromosomal aberrations was higher in DNA aneuploid tumors than in diploid tumors (8.17 vs 3.60/tumor, P<0.05). Furthermore, the average number of chromosomal aberrations was significantly higher in stage T2 tumors and larger tumors than in stage T1 tumors (7.71 vs 3.25/tumor, P<0.05). Our results suggest that DNA aneuploidy and large tumor size reflect an underlying chromosomal instability.

Identification of genetic aberrations in cell lines from oral squamous cell carcinomas by comparative genomic hybridization.

We detected genetic alterations in 14 cell lines established from 14 human oral squamous cell carcinomas (OSCCs) using comparative genomic hybridization (CGH), which allows a comprehensive analysis of chromosomal imbalances and identification of nonrandom genetic aberrations specific to OSCCs. All cell lines showed gains and losses of DNA copy number. DNA losses were detected for chromosomes 18q (10/14) and 4q (9/14) with minimal overlapping regions of 18q12-32 and 4q31-qter, respectively. In contrast, the common sites for increased copy number were chromosomes 5p (12/14), 8q23-ter (11/14), 20p (8/14), 20q (8/14), and 3q25-ter (7/14). These results suggest that losses of 18q12-22 and 4q31-ter and gains of 5p and 8q23-ter play important roles in the development and/or progression of OSCC.

Chromosome 17 abnormalities in squamous cell carcinoma of the oral cavity, and its relationship with p53 and Bcl-2 expression.

Numerical aberrations of chromosome 17 were studied by fluorescence in situ hybridization (FISH) using pericentromere specific DNA probe in 27 oral squamous cell carcinomas (SCCs), and its relationship with p53 and Bcl-2 protein expression was investigated. Since cells with polysomy 17 are significantly increased in SCC (p = 0.0005), chromosome 17 abnormality seems to be correlated with carcinogenesis of oral SCC. Chromosome 17 abnormality varied from case to case, and the degree of the abnormality did not correlate with the stage of the disease, the histological differentiation of SCC, or relapse of the disease and lymph node metastasis. However, there was a correlation between polysomy 17 and p53 immunoreactivity (p = 0.0228). p53 immunoreactivity showed a correlation with relapse of the disease (p = 0.0441). An inverse relationship was found between p53 and Bcl-2 immunoreactivity (p = 0.0421). In conclusion, we suggest that polysomy 17 is closely related carcinogenesis of oral SCC, and with p53 overexpression. It is assumed that polysomy 17 correlates with the mutation of p53 which results in an accumulation of aberrant p53 protein, and that its activation causes an increase of p53 protein.

Conformational change of ascidiacyclamide caused by asymmetric modification for an isoleucine residue: structural analyses of [Gly], [Leu], and [Phe]ascidiacyclamides by x-ray diffraction and NMR spectroscopy.

Ascidiacyclamide, a cytotoxic cyclic peptide from tunicate, is composed of unusual amino acids and has a repeated sequence, c[-thiazole-D-Val-oxazoline-L-Ile-]2 ([Ile]ASC). The symmetric chemical structure has been assumed to be correlated with the cytotoxicity, and it is reasonable to consider that the disturbance of its structure from the C2 symmetry results in the changes of conformation and activity. In order to quantitatively estimate the molecular conformation-activity relationship, an isoleucine residue was substituted by Gly, Leu, or Phe to disturb the C2 symmetry. The conformations of three derivatives were examined by nmr spectroscopy and the crystal structure of [Leu]ASC was also analyzed by x-ray diffraction method. The 1H-nmr experiments and the constrained molecular dynamics simulations showed the twisted "figure 8" conformers for [Gly] and [Phe]ASCs and the "square" conformer for [Leu]ASC in the DMSO solution. The x-ray crystal analysis of [Leu]ASC also revealed the square form similar to the solution structure. On the other hand, their cytotoxic activities were measured using L1210 leukemia cells and were related with the bulkiness and/or hydrophobicity of the side chain of the substituted amino acid; [Phe] > or = [Ile] > [Leu] >> [Gly]ASCs. As an attempt to consider the correlation between the activity and conformer, the accessible surface area (ASA) was calculated for each derivative to estimate the size or bulkiness of its conformation. Although the ASAs of nmr structures were not directly related to the type of conformer (figure 8 or square form), it was an important probe to consider the cytotoxicity of each derivative.

Detection of Epstein-Barr virus in oral papilloma.

Fifty-one cases of malignant and non-malignant oral diseases were investigated for Epstein-Barr virus (EBV). EBV DNA was detected by polymerase chain reaction analysis in 2 of 4 papillomas, but not in other tissues including 36 squamous cell carcinomas and 4 leukoplakias. The copy numbers of EBV DNA in the two positive samples were estimated to be 120 and 36 per cell, respectively. Intense EBV DNA signals were detected on papilloma cells by in situ hybridization. DNAs for the benign and malignant types of human papilloma virus were not detected in papilloma tissues. The present results suggest that EBV is a causative agent of oral papilloma.

Expression of ICAM-1 in implanted primary and metastatic squamous cell carcinomas in rats.

FF6 tumor cells are derived from a spontaneous rat squamous cell carcinoma (SCC) which originally arose in the facial skin of a DA rat. In this study, FF6 tumor cells were implanted into rat oral mucosa to establish an ex vivo metastatic model. We analyzed the expression of intercellular cell adhesion molecule-1 (ICAM-1) in the implanted primary and metastatic FF6 tumors by immuno-staining with a monoclonal antibody (mAb) against ICAM-1. The implanted primary FF6 cells showed strong expression of ICAM-1, whereas the tumor cells of metastatic lesions showed weak or negative expression of ICAM-1. By immunostaining with mAb OX6, a number of MHC class II-positive macrophages were detected in tumor mesenchyme and surrounding the metastatic foci. These results suggested that the local immune reaction in the lymph node influenced the expression of ICAM-1 on tumor cells, and that MHC class II-positive macrophages may play a role in transplanted tumor growth and metastases.

Detection of numeric abnormalities of chromosome 17 and p53 deletions by fluorescence in situ hybridization in pleomorphic adenomas and carcinomas in pleomorphic adenoma. Correlation with p53 expression.

BACKGROUND: A fluorescence in situ hybridization (FISH) technique using specific DNA probes allows for the detection of chromosomal aberrations and gene deletions and gains, even in interphase nuclei in human solid tumors. A high frequency of aberrations of chromosome 17 and mutation of the p53 gene have been reported in some human tumors. The correlation of p53 expression with abnormalities of chromosome 17 and p53 gene deletion in salivary gland tumors has not yet been investigated. METHODS: The authors analyzed the numeric aberrations of chromosome 17 and p53 gene deletions in 11 paraffin embedded pleomorphic adenomas (PA) and 9 carcinomas in pleomorphic adenoma (CIPA), using FISH techniques. The centromere specific DNA probe for chromosome 17 and p53 cosmid DNA probe was used. The aberrations of chromosome 17 and p53 deletion were correlated with immunohistochemical detection of p53 protein. RESULTS: Monosomy 17 was detected in 30.8% of CIPA cells and 29.6% of PA cells, and polysomy 17 was detected in 19.6% of CIPA cells and 9.6% of PA cells. p53 protein expression was observed in 6 of 9 CIPA specimens (66.7%) and 2 of 75 PA specimens (2.7%). Deletion of the p53 gene was frequent in p53 protein positive specimens. A statistically significant correlation existed between p53 protein expression and polysomy 17 (P = 0.0417). CONCLUSIONS: It was observed that loss of chromosome 17 may occur in PA before its transformation to carcinoma. p53 expression was frequently associated with deletion of the p53 gene as detected by FISH. Polysomy 17 was more frequent in CIPA than PA and was associated with mutation of p53.

Epstein-Barr virus (EBV) infection in salivary gland tumors: lytic EBV infection in nonmalignant epithelial cells surrounded by EBV-positive T-lymphoma cells.

To elucidate the association of Epstein-Barr virus (EBV) and salivary gland tumors, 114 cases of tumors of major salivary glands were investigated. EBV DNA was detected in all 6 cases of undifferentiated carcinoma and all 3 cases of T-cell lymphoma, but not in other tumor tissues. In situ hybridization studies for EBV DNA and EBV-encoded small RNA 1 (EBER1) showed specific localization of the EBV sequences to the undifferentiated carcinoma cells and T-lymphoma cells. Moreover, intense DNA signals were detected on nonneoplastic epithelial cells of T-lymphoma tissues. These epithelial cells were negative for EBER1 and expressed BZLF1, BALF2, and gp350/220 proteins associated with virus production. In contrast, nonmalignant epithelial cells surrounded by undifferentiated carcinoma cells showed no evidence of EBV infection or virus replication. These results indicate that there is an unusual association of salivary gland T-cell lymphomas with lytic EBV replication of nonmalignant epithelial cells.

Detection and analysis of human papillomavirus 16 and 18 homologous DNA sequences in oral lesions.

The prevalence of human papillomavirus (HPV) 16 and 18 was investigated in oral lesions of the population of northeast China including squamous cell carcinomas (SCCs), candida leukoplakias, lichen planuses and papillomas, by southern blot hybridization with polymerase chain reaction (PCR). Amplified HPV16 and 18 E6 DNA was analyzed by cycle sequence. HPV DNA was detected in 14 of 45 SCCs (31.1%). HPV18 E6 DNA and HPV16 E6. DNA were detected in 24.4% and 20.0% of SCCs. respectively. Dual infection of both HPV 16 and HPV 18 was detected in 6 of 45 SCCs (13.3%), but not in other oral lesions. HPV 18 E6 DNA was also detected in 2 of 3 oral candida leukoplakias, but in none of the 5 papillomas. Our study indicated that HPV 18 infection might be more frequent than HPV 16 infection in oral SCCs in northeast Chinese, dual infection of high risk HPV types was restricted in oral SCCs, and that HPV infection might be involved in the pathogenesis of oral candida leukoplakia.

Recognition of a special membrane antigen of squamous cell carcinoma in rats with a monoclonal antibody UB23.

This article describes the recognition of a special membrane antigen of the rat squamous cell carcinoma (SCC) by a monoclonal antibody (mAb), UB23, and the characterization of the UB23 antigen expression in the implanted primary and metastatic SCC in rat models. The mAb UB23 was raised against the FF6 tumor, a well-differentiated rat SCC, and it recognized the 120- to 130-kD cell surface antigen in FF6 tumor cells. The UB23 antigen was found in frequently observed small 'basal' cells but not in keratinocytes, and an increased expression was seen in the cells at the interface with peritumoral stroma in both the implanted primary FF6 tumors and metastases. These results indicated that the UB23 antigen is closely related with the cell differentiation and invasion of FF6 cells, and could be useful for analyzing the mechanism of differentiation, invasion and metastasis of SCC in animal models.

Increased polysomy 3 and 17 detected by fluorescence in situ hybridization (FISH) in oral squamous cell carcinoma.

Numerical chromosome aberrations were studied by fluorescence in situ hybridization (FISH) using pericentromeric DNA probes specific for chromosomes 3 and 17 in 18 oral squamous cell carcinomas (SCCs) and 3 lymph nodes as control. Disomy 3 and 17 was detected in approximately 90% of control cells, and in 60.9 +/- 4.0% and 62.7 +/- 3.6% of SCCs, respectively. Polysomy 3 and 17 significantly increased in SCCs when compared to controls. The pattern of chromosome aberrations varied considerably between cases. There was no obvious relationship between the degree of polysomies and clinicopathological factors such as tumor-differentiation, stage of the disease and clinical outcome. Chromosome aberrations by FISH did not correlate with DNA aneuploidy by flow cytometry. Our results indicate that oral SCCs are more frequently associated with the increased copy number of chromosomes 3 and 17 than previously thought, and that a correlation between chromosome aberrations by FISH and DNA aneuploidy by flow cytometry is not obvious.

Association of Epstein-Barr virus (EBV) with Sjögren's syndrome: differential EBV expression between epithelial cells and lymphocytes in salivary glands.

The association of Epstein Barr virus (EBV) with Sjögren's syndrome (SS) is still in dispute. This study is aimed to investigate the existence of EBV genomes and their products in salivary glands of SS. Salivary gland samples were surgically obtained from Chinese patients. EBV DNA was detected in three of seven cases by dot blot hybridization and in four of seven cases by in situ hybridization. The EBV-encoded small RNA-1 (EBER1) was detected in two of seven cases by in situ hybridization. The immunohistochemical staining of EBV proteins showed that the EBV latent membrane protein-1 was detected in four of seven cases and that BZLF1, BALF2, and gp350/220 proteins associating with virus production were not expressed. In eight controls, no positive signal was observed by these methods. DNA in situ hybridization identified ERV on both epithelial cells and lymphocytes. On the other hand, EBER1-positive signals were exclusively localized on lymphocytes. These results indicate that two forms of EBV infection may exist in salivary glands of SS. One is EBER1-positive latency in lymphocytes, the other is EBER1-negative latency in epithelial cells. Frequent EBV detection in salivary glands of SS suggests that EBV plays a role in the genesis of SS.

[Hereditary gingival fibromatosis].

Fibromatosis may be defined as diffuse poorly circumscribed overgrowth of the fibrous tissue that infiltrates adjacent normal tissues. They are difficult to eradicate surgically, and recur but not metastasize. Gingival fibromatosis is generally regarded as a disease that leads to an extensively diffuse and remarkable hyperplasia of the maxillo-mandibular gingiva. Occasionally, this lesion covers all teeth. The histogenesis of the fibromatosis remains unexplained. Trauma, endocrine, idiopathic factors and genetic factors have been implicated, but it is uncertain whether any of then play a major role in the development of the disease. Occasional cases with familial history have been reported. The treatment of choice would appear to been block resection of the tumor and surrounding normal structures. Although, this lesion has a high recurrent rate. For this reason, in many of the case reports, that it has been recommended that the follow up period is considerably less than three years.

A fluorescence in situ hybridization (FISH) analysis with centromere-specific DNA probes of chromosomes 3 and 17 in pleomorphic adenomas and adenoid cystic carcinomas.

Aberrations of chromosomes 3 and 17 were studied by FISH using centromere-specific DNA probes in 11 salivary adenoid cystic carcinomas (ACC) and 8 salivary pleomorphic adenomas (PA), with 3 lymph nodes as controls. Two hybridized signals were detected in 92.8 +/- 2.7% of controls, 73.2 +/- 7.0% of PA and 66.8 +/- 7.9% of ACC cells for chromosome 3, and in 90.4 +/- 2.3% of controls, 59.5 +/- 25.0% of PA and 44.8 +/- 20.2% of ACC for chromosome 17. More than 3 hybridized signals, which indicate polysomy, were observed in 3.1% of controls, 15.5% of PA and 22.9% of ACC cells for chromosome 3, and in 1.2% of controls, 10.3% of PA and 23.1% of ACC cells for chromosome 17. A single hybridized signal was much more frequent for chromosome 17 than for chromosome 3. These findings suggest that polysomy of both chromosomes occurs during the development of salivary gland tumors, and its frequency is increased in adenoid cystic carcinoma as compared to pleomorphic adenoma. In addition, monosomy of chromosome 17 could possibly be significant in salivary gland tumors.

The significance of PCNA and p53 protein in some oral tumors.

The expression of PCNA and p53 protein was evaluated in a total of 75 cases of benign and malignant lesions of the oral cavity, comprising 50 squamous cell carcinomas (SCCs), 14 leukoplakias, and 11 pleomorphic adenomas. The DNA histogram of 20 SCCs was measured by flow cytometry. p53-positive cells were frequently seen in SCCs, but were rare in leukoplakias and pleomorphic adenomas. The PCNA labeling index (LI) was higher in SCCs than in other benign lesions. The expression rate of p53 protein was markedly elevated in SCCs obtained from smoking patients, when compared to nonsmoking patients. DNA ploidy did not show a close relationship with PCNA and p53 expression. The mean value of PCNA LI for 22 cases carrying positive p53 protein was 52.3%, which was higher than that of p53 protein negative cases (35.7%). The Kaplan-Meier survival curve of the patients who were negative for p53 was significantly more favorable than for patients who were positive for p53 (P < 0.01, Cox-Mantel test). These results suggest that PCNA and p53 LI are markers for the malignant potential of the oral mucosa, and are a useful indicator suggesting a poor prognosis.

Cytoplasmic expression of p53 protein and its morphological features in salivary gland lesions.

An immunohistochemical study of p53 protein was carried out on 45 salivary gland lesions using a monoclonal antibody, Bp53-12, raised to the intracellular domain of the p53 protein. p53 protein expression was found in 34.4% of 32 salivary gland carcinomas. Nuclear p53 expression was detected in tumor cells but not in non-neoplastic cells, except in one salivary duct carcinoma. The perinuclear cytoplasm of luminal duct cells was specifically positive for the antibody used here. Cytoplasmic p53 expression was observed mostly in non-neoplastic cells. There was a tendency for the cytoplasmic staining of p53 protein to be observed in the normal cells adjacent to p53-positive carcinomas, but none of the normal cells were positive in the tissues surrounding p53-negative carcinomas. Cytoplasmic expression of p53 protein in salivary gland tissues seems to be correlated with tumorigenesis.

Immunohistochemical detection of p53 protein and proliferating cell nuclear antigen (pcna) in salivary-gland tumors.

The expression of p53 protein and PCNA was immunohistochemically examined in 161 cases of salivary gland tumors. The p53 protein was positive in 23.8% (24/101) of malignant salivary gland tumors, while only one case of 60 pleomorphic adenomas was positive. p53 protein was frequently detectable in salivary duct carcinoma (60%), basal cell adenocarcinoma (66.7%), acinic cell carcinoma (53.3%) and undifferentiated carcinoma (50%), but was rarely seen in adenoid cystic carcinoma (17.2%) and mucoepidermoid carcinoma (0%). p53 protein was more frequently detectable in PCNA positive cases than in negative cases (p=0.0074).