Differential expression of stem cell-like proteins in normal,hyperplastic and dysplastic oral epithelium

Objective

The identification of stem cells (SC) remains challenging. In the human oral mucosal epithelium, these cells are believed to be in the basal layer (stem cell niche), but their exact location is unclear. The aim of this study was to examine the dysplastic oral epithelium for these SC-like proteins in order to assess their diagnostic value as biomarkers complementing the histological grading of dysplasia.

Material and Methods

Thirty oral epithelial dysplasia (OED), 25 oral lichen planus (OLP), 10 oral hyperkeratosis and 5 normal oral epithelium (OE) were immunohistochemically examined for four SC markers [integrin β1, neuron-glial-2 (NG2), notch 1 (N1) and keratin 15 (K15)].

Results

Three of four SC markers were heterogeneously detected in all samples. K15 overexpression in the lower two-thirds of severe OED suggests an expanded SC niche. Integrin β1 distribution pattern was not measurably different between OEDs and control. NG2 was almost negative to absent in all samples examined. N1 expression was weak and highly variable in normal and dysplastic epithelium, making it an unreliable epithelial stem cell marker.

Conclusions

Present findings suggest that these markers were unable to identify individual epithelial stem cells. Instead, subpopulations of cells, most probably stem cells and transit amplifying cells with stem cell-like properties were identified in the dysplastic oral epithelium. The characteristic expressions of K15 might be of diagnostic value for oral dysplasia and should be investigated further.     

P53蛋白在口腔鳞状细胞癌发生过程中的表达及意义

本研究采用微波处理暴露抗原,通过免疫组化ＬＳＡＢ方法对ＮＯＭ６例,ＯＰＬ２８例,ＯＳＣＣ３４例中Ｐ５３蛋白表达进行观察,其ＮＯＭ无表达;在ＯＰＬ中的表达率为８２％随着上皮异常增生的程度加重,表达率呈上升趋热（Ｐ〈０．０５）;而在ＯＳＣＣ中表达率为６５％,随着肿瘤的分极增加,表达率呈下降趋势（Ｐ〈０．０５）。结果表明,Ｐ５３蛋白在ＯＰＬ和ＯＳＣＣ中呈异常表达,并与ＯＰＬ程度和ＯＳＣＣ分级有关,Ｐ５３     

The permeability of hyperplastic oral epithelium

An epithelial hyperplasia is one of the reactions of skin and oral mucosa to chemical and mechanical insult. It is usually assumed that this reaction produces a more effective epithelial barrier, but there is no information as to whether a less permeable tissue results. To examine this question, hyperplasia was induced in the cheek pouches of hamsters by either chemical treatment with 0.0025% TPA in acetone or by mechanical abrasion with a rotating mop; untreated hamsters served as controls. The animals were killed and the cheek pouches were removed, mounted in diffusion chambers and the permeability to labelled water and horseradish peroxidase (HRPO) determined. The results showed that higher values were obtained for the permeability constant of hyperplastic epithelia than for that of control tissue, suggesting that an increased epithelial thickness is not necessarily associated with an improved permeability barrier function. The presence of an inferior barrier layer in hyperplastic epithelia may be related to the increased rate of turnover of this tissue.     

The permeability of hyperplastic oral epithelium

An epithelial hyperplasia is one of the reactions of skin and oral mucosa to chemical and mechanical insult. It is usually assumed that this reaction produces a more effective epithelial harrier, but there is no information as to whether a less permeable (issue results. To examine this question, hyperplasia was induced in the cheek pouches of hamsters by either chemical treatment with 0.0025% TPA in acetone or by mechanical abrasion with a rotating mop; untreated hamsters served as controls. The animals were killed and the cheek pouches were removed, mounted in diffusion chambers and the permeability to labelled water and horseradish peroxidase (IIRPO) determined. The results showed that higher values were obtained for the permeability constant of hyperplastic epithelia than for that of control tissue, suggesting that an increased epithelial thickness is not necessarily associated with an improved permeability barrier function. The presence of an inferior barrier layer in hyperplastic epithelia may be related to the increased rate of turnover of this tissue.     

Integrin expression in oral hairy leukoplakia and normal tongue epithelium

OBJECTIVE: To describe the expression of integrins in the epithelium of oral hairy leukoplakia (HL) and compare to that of normal lateral tongue epithelium. MATERIALS AND METHODS: Immunohistochemistry to identify integrins (alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1) was performed, using a standard biotin-streptavidin-peroxidase technique on five clinically and histologically confirmed frozen biopsy specimens of HL and five normal lateral tongue control tissues. RESULTS: Expression of integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 was seen both in HL epithelium and in normal control tissue. alpha 5 expression was not seen in HL or in control tissue epithelium. alpha 2 and alpha 3 were expressed mainly in the basal and suprabasal layers; alpha 6 expression was most intense on the basal surface of the basal cells, alpha v was expressed in the basal and suprabasal layers with more expression seen in the higher differentiated cell layers than the other integrins. beta 1 expression was seen in the basal and suprabasal layers only. No apparent difference between HL and normal oral mucosa was noted in the staining pattern of the various integrins. CONCLUSION: Integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 are expressed in HL and the expression pattern is not different from that of normal oral mucosa. alpha 5 is not expressed in HL or in normal oral epithelium.     

人牙龈结合上皮、口腔龈上皮及沟内上皮中细胞角蛋白的表达

目的 检测人牙龈上皮的细胞角蛋白(CK)谱，探讨结合上皮(JE)与口腔龈上皮(OE)、沟内上皮(SE)的不同。方法 5例人磨牙及前磨牙牙龈颊舌向石蜡切片，采用免疫组化SP法行CK5／6、7、8／18、10／13、16、17、19、20染色。结果 CK7、17在3种上皮中均无表达；CK5／6、20在3种上皮的基底上层(尤其是近表层)为弱阳性及阳性表达，基底层无表达；CK10／13、16在JE全层均有表达，在OE和SE仅限于基底上层，其中CK10／13为强阳性，CKl6为弱阳性及阳性；CK19在JE全层强阳性，在OE和SE仅限于基底层，JE和SE分界明显；CK8／18与CK19相似，只是表达较弱，近基底层的基底上层也有少量表达。结论JE是一种不同于OE和SE的特殊低分化上皮，CK19可作为JE的特征性标记物区别于OE和SE，CK10／13、16可用于体外培养的上述3种细胞的鉴别。     

Plasminogen activators in normal and malignant oral epithelium in vivo and in vitro.

Urokinase-type (uPA) and tissue-type (tPA) plasminogen activators were identified by fibrinolytic autography in the sulcus epithelium of human gingival mucosa but not in the orthokeratinized gingival epithelium. Fibrinolytic activity was present only over blood vessels in frozen sections of oral squamous cell carcinomas, the malignant epithelial cells showing no plasminogen activator activity. Plasminogen activators could not be demonstrated in either the sulcus or gingival epithelium by immunofluorescence, but both uPA and tPA were found in occasional squamous carcinoma cells. Fibrinolytic activity of culture fluids from epithelial explants grown in vitro from human gingival mucosa showed marked variation, but activity was much higher in the culture supernatants than in the cell lysates. Fibrinolytic activity of culture fluids from epithelial explants of squamous cell carcinomas was low both in supernatants and lysates. Zymogram overlays of sodium dodecyl sulphate-polyacrylamide electrophoretic gels from culture supernatants showed that the low fibrinolytic activity of culture supernatants of oral squamous cell carcinomas was due to the associated presence of plasminogen activator inhibitors. The fibrinolytic activity in the zymogram was due predominantly to uPA but some lysis was due also to tPA.     

RAS oncogene product expression in normal and malignant oral mucosa

Proto-oncogenes are important in both normal cellular differentiation and in carcinogenesis. The majority of transforming genes belong to the ras family and the ras gene product has been shown to be elevated in some oral carcinomas. RAP-5 monoclonal antibody was used to determine the expression of the p21ras protein in normal and neoplastic oral mucosa in an immunohistological study. The expression of p21ras protein was generally restricted to acanthous cells with strong staining in normal oral mucosa and well-differentiated carcinomas. In contrast, the p21ras protein was not detected in significant amounts in severely dysplastic lesions and poorly differentiated carcinomas. These results suggest that expression of p21ras is a normal feature of more fully differentiated tissues, both normal and neoplastic, and is not useful as an indicator of cell proliferation or 'malignant potential'.     

Detection of human papillomavirus-16 and HPV-18 DNA in normal,dysplastic, and malignant oral epithelium

OBJECTIVE: Our aim was to clarify the association of human papillomavirus (HPV) with oral carcinogenesis, especially its early stage. STUDY DESIGN: Tissue specimens of normal mucosa, epithelial dysplasia, oral squamous cell carcinoma (OSCC), and OSCC cell lines were examined for the presence of HPV-16 and HPV-18 E6 DNA by means of the polymerase chain reaction test. RESULTS: The detection rate of HPV-16 in epithelial dysplasia (31/51) was higher than that in normal mucosa (16/44) and in OSCC (30/86) and was statistically different from that in OSCC. The cases that progressed from epithelial dysplasia to carcinoma showed a significantly higher HPV-16 detection rate than the other cases in both epithelial dysplasia and OSCC. HPV-16 and HPV-18 were detected only at early passages of 2 of 10 OSCC cell lines. CONCLUSIONS: These results strongly suggest that HPV-16 may be involved in the early stages of the development of some oral carcinomas.     

Stromal laminin chain distribution in normal,hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation

J Oral Pathol Med (2010) 39 : 290–298 Background: The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains α2, α3, α4, α5 and γ2 in the stromal compartment/vascular structures in OSCC was analysed. Methods: Frozen tissue of OSCC (9× G1, 24× G2, 8× G3) and normal (2×)/hyperplastic (11×) oral mucosa was subjected to laminin chain and α‐smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. Results: Stromal laminin α2 chain significantly decreases and α3, α4, α5 and γ2 chains and also ASMA significantly increase with rising grade. The amount of stromal α3, α4 and γ2 chains significantly increased with rising ASMA positivity. There is a significant decrease in α3 chain positive vessels with neoplastic transformation. Conclusions: Mediated by myofibroblasts, OSCC development is associated with a stromal up‐regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning α3 and γ2 chain laminins during tumour angioneogenesis is suggested.     

Epstein-Barr virus receptors but not viral DMA are present in normal and malignant oral epithelium

The presence and distribution of Epstein-Barr Virus receptors (EBVR's) on a range of normal (n = 18), dysplastic (n = 10) and malignant (n = 20) oral mucosa were studied by immunocytochemical methods using the monoclonal antibodies (MAb's) HB5 and B2. EBVR's were demonstrated as membrane staining of the spinous layers of normal non- and parakeratinized epithelium, indicating that EBVR's are differentiation-linked. This distribution was retained in dysplastic epithelium. Tissue from oral squamous cell carcinomas (SCC's) showed variable reactivity of only a few cells scattered randomly within the samples. Furthermore, a sensitive in situ hybridization (ISH) technique was used to determine if Epstein-Barr virus (EBV) was present in normal (n = 15) and oral squamous cell carcinoma tissue (n = 20). No EBV DNA was demonstrated within either normal or malignant epithelium, suggesting that the virus does not persist in normal oral stratified squamous epithelium nor is there any evidence for a role in oral carcinogenesis.     

Apoptosis in normal epithelium,premalignant and malignant lesions of the oropharynx and oral cavity: a preliminary study

To explore the involvement of apoptosis in the development of oral and oropharyngeal squamous cell carcinoma (SCC) in vivo, biopsies were taken from patients with macroscopically normal (n = 6), leukoplakic (n= 12) or malignant (n = 8) mucosa. Leukoplakic lesions were divided histologically into dysplasia (n=5) or carcinoma in situ (CIS: n=7). Material was prepared for light and electron microscopy. The apoptotic index (AI), vertical cell position of apoptoses (cp), mitotic index (MI) and AI:MI ratio were calculated for each patient. AI increased from 0.12% ± 0.07 S.E.M. (normal) to 0.58 ± 0.13 (CIS) but fell to 0.14 ± 0.14 in SCC. Apoptoses were suprabasal in normals, but generalised in CIS. MI increased from normal (0.20 ± 0.06) to SCC (0.32 ± 0.09), and AI:MI was at its maximum in CIS (1.57; SCC: 0.44). The results suggest that a change in apoptosis accompanies the onset of invasion in a premalignant lesion of the human oral cavity and oropharynx.     

Difference in glycogen metabolism (glycogen synthesis and glycolysis) between normal and dysplastic/malignant oral epithelium

BackgroundThe purpose of this study was to investigate a difference in glycogen metabolism (glycogen synthesis and glycolysis) between the iodine stained (normal non-keartinized) and the unstained (dysplasctic/malignant) oral epithelium.MethodsTwenty-one frozen tissue samples of iodine-stained and unstained mucosal tissue were obtained from 21 OSCC patients. Serial frozen sections were cut and examined with the hematoxylin-eosin and periodic acid-Schiff methods and immunohistochemical (IHC) staining for Ki67, P53, molecules associated with glycogenesis (i.e., glycogen synthase (GS) and phospho-glycogen synthase (PGS)), and molecules associated with glycogenolysis (i.e., glycogen phosphorylase isoenzyme BB (GPBB) examine the glycogen metabolism in OSCC. Additionally, in vitro study, the expression levels of GS and GPBB in the cultured cells were analyzed by immunofluorescent staining, Western blot analysis, and the real-time quantitative polymerase chain reaction (PCR).ResultsThere was no significant difference in GS and PGS immunoactivity between iodine stained and unstained area. On the other hand, significantly greater GPBB immunoreactivity was observed in the basal and parabasal layers of iodine-unstained epithelium, where higher positivity for p53 and Ki67 was also showed. Additionally, western blot analysis, immunofluorescent staining, and real-time quantitative PCR revealed that the oral squamous cancer cells exhibited greater expression of GPBB than normal epithelial cells.ConclusionsThe results of this study showed that GPBB expression, which resulted in up-regulation of glycogenolysis, is enhanced in oral dysplastic/malignant epithelium compared with non-keartinized normal epithelium, in spite of the fact that glycogenesis continues in both of them. Premalignant and malignant epithelial cells consume greater quantities of energy due to their increased proliferation, and hence, exhaust their glycogen stores, which resulting in negative stain reaction with iodine solution.     

Differential expression of E-cadherin and cytokeratin 19 and net proliferative rate of gingival keratinocytes in oral epithelium in periodontal health and disease

BACKGROUND: The effects of periodontal disease on the oral gingival epithelium (OGE) have not been documented fully because they may not be as dramatic as those seen on the junctional epithelium. The aim of this study was to estimate the changes occurring in the OGE with respect to its proliferation and E-cadherin and cytokeratin 19 (K19) expression during pocket formation. METHODS: Gingival samples were collected from 17 periodontally healthy subjects and 18 subjects with chronic periodontitis. K19 and E-cadherin levels were analyzed immunohistochemically. The net proliferative rate was calculated as the difference between the proliferative rate and the apoptotic rate as determined by immunohistochemical analysis of Ki-67 and p53, respectively. RESULTS: There was a significant increase in the net proliferative rate of the OGE during pocket formation (periodontitis group, 220.90+/-46.85; healthy group, 107.60+/-25.86; P<0.001). There was a significant reduction in E-cadherin expression (periodontitis group, 0.837+/-0.428; healthy group, 1.846+/-0.555) and a significant increase in K19 expression during pocket formation (periodontitis group, 1.45+/-0.686; healthy group, 0.533+/-0.410). CONCLUSION: OGE appears to undergo significant changes in proliferation and differentiation during pocket formation that do not seem to be restricted to proteolytic destruction by the invading microorganisms.     

Prevalence of p53, bcl-2, and Ki-67 immunoreactivity and of apoptosis in normal oral epithelium and in premalignant and malignant lesions of the oral cavity.

PURPOSE: Loss of normal p53 is correlated to the progression of several preneoplastic lesions to neoplasms, and overexpression of bcl-2 determines an alteration of programmed cell death. There is an increased awareness of the importance of apoptosis in cancerogenesis, and a strong correlation of Ki-67 with high tumor grade has been reported. MATERIALS AND METHODS: The aim of our study was to investigate immunohistochemically the expression and relationship of p53, bcl-2, MIB-1, and the apoptotic index (AI) in normal oral epithelium, leukoplakia, dysplasia, and oral squamous cell carcinoma. RESULTS: A strong correlation was found between p53 overexpression and cell proliferation (MIB-1) and the AI. An inverse relationship was found between bcl-2 expression and MIB-1 and AI. A significant inverse relationship was found between p53 and bcl-2. A good positive correlation was present between AI and MIB-1 expression. CONCLUSIONS: Apoptosis could be important to help to understand oral carcinogenesis.     

Antioxidant enzyme levels in oral squamous cell carcinoma and normal human oral epithelium

BACKGROUND: The antioxidant enzymes (manganese- and copper-zinc-containing superoxide dismutases, catalase and glutathione peroxidase) limit cell injury induced by reactive oxygen species. The purpose of the study was to determine whether human oral squamous cell carcinomas have altered antioxidant enzyme levels. This study is the first to undertake this task in human oral mucosa and squamous cell carcinoma. METHODS: Semiquantitative immunohistochemistry was used to examine 26 archived oral squamous cell carcinoma biopsies. Fourteen well-differentiated and 12 poorly differentiated tumors were examined, as were 12 specimens of oral mucosa. All sections were reviewed by two oral and maxillofacial pathologists, and image analysis of the immunostained sections was performed using NIH Image. Antioxidant enzyme staining intensities were compared in the different groups by Duncan's multiple range test. RESULTS: In general, mucosal basal cells displayed lower antioxidant enzyme levels than spinous cells, and primary tumor cells displayed lower antioxidant enzyme staining intensities than did their normal cell counterparts. Moreover, poorly differentiated tumor cells showed lower antioxidant enzyme staining intensities than well-differentiated tumor cells. Manganese-containing superoxide dismutase staining intensities were, however, higher in well-differentiated oral squamous cell carcinomas than their normal cells of origin. CONCLUSIONS: Detection of antioxidant enzymes may be a useful future marker in the molecular diagnosis of the oral cancer. Moreover, it may be possible to not only monitor the effectiveness of chemopreventive and therapeutic strategies in oral cancer using these enzymes, but to monitor tumor recurrence.     

LETS protein in normal and pathological human oral epithelium.

The distribution of LETS protein in human oral mucosa was studied by indirect immunofluorescence. Normal epithelium showed surface staining. Intracellular staining occurred in epithelial cell cytoplasm in lichen planus and pemphigoid. Surface staining was absent in discoid lupus erythematosus. In pemphigus, intercellular staining was seen near areas of acantholysis.