Oral Leukoplakia Cell Research Articles

Lytic Epstein–Barr virus infection in epithelial cells but not in B-lymphocytes is dependent on Blimp1

Epstein-Barr virus (EBV) replicates in superficial differentiated cells of oral hairy leukoplakia (OHL). Differentiation of squamous epithelial cells depends on B-lymphocyte-induced maturation protein 1 (Blimp1). Here we show that expression of the EBV immediate-early protein BZLF1 is restricted to Blimp1-positive epithelial cells in OHL. Luciferase assays revealed Blimp1-dependent induction of the BZLF1 promoter Zp in epithelial cell lines. Expression of ZEB1, a negative regulator of Zp, and of Xbp-1, which mediates the Blimp1 effect on Zp in B-cells, was not affected by enforced Blimp1 expression. Moreover, Xbp-1 protein expression was not detected in differentiated epithelial cells of OHL. Thus, Blimp1 induces BZLF1 expression in epithelial cells independently of ZEB1 and Xbp-1. In contrast to epithelial cells of OHL, BZLF1 expression was also observed in Blimp1-negative lymphoid cells in infectious mononucleosis tonsils, suggesting that EBV replication in B-cells may be induced independently of terminal differentiation.

Immunohistochemical evaluation of bcl-2 oncoprotein in normal oral mucosa, leukoplakia and oral squamous cell carcinoma

Immunohistochemical evaluation of bcl-2 oncoprotein in normal oral mucosa, leukoplakia and oral squamous cell carcinoma

Diagnostic Reliability of Fine Needle Aspiration Cytology against Histopathology for the Diagnosis of Oral Squamous Cell Carcinoma and Oral Leukoplakia

The purpose of this study was to determine the possibility of using fine needle aspiration cytology (FNAC) as a primary diagnostic test in oral leukoplakia and squamous cell carcinoma. This study consisted of clinically diagnosed 15 cases of leukoplakia and 15 cases of oral squamous cell carcinomas. FNAC and biopsy were done on all the cases. A cytological and histopathological correlation was undertaken to determine the proportion of cancers. A 23-gauge sterile disposable needle was attached to a disposable syringe and introduced into the lesion at the proposed biopsy site in one movement. In leukoplakias, the center of the lesion or erythroplakic areas and, in squamous cell carcinomas, proliferative areas and edges of the ulcers were chosen. In leukoplakia group, out of 15 biopsy samples, one (6.67%) sample was negative and 14 (93.33%) were positive. Whereas out of 15 FNAC samples, 14 (93.33%) were negative and one (6.67%) sample was positive. In squamous cell carcinoma, out of 15 biopsy samples, no sample was negative and all (100.00%) were positive. Whereas out of 15 FNAC samples, two (13.33%) were negative and 13 (86.67%) sample were positive. It is noted that FNAC can be employed as a sound diagnostic tool for rapid diagnosis of oral squamous cell carcinoma. It may be particularly useful in cases, where formal biopsy procedure is difficult or contraindicated due to medical reasons or in cases of advanced malignancy. FNAC has been shown to be reliable and safe technique in the diagnosis of malignant in the head and neck. When the aspirations are performed by cytopathologists, it is easy to perform a rapid staining of the first smear and within 10 to 15 minutes to ensure that the material is sufficient and diagnosable and to suggest a preliminary diagnosis.

O63. Comparison of degranulated mast cell counts in oral leukoplakia and oral squamous cell carcinoma

O63. Comparison of degranulated mast cell counts in oral leukoplakia and oral squamous cell carcinoma

Analysis of cell proliferation rate in Oral Leukoplakia and Oral Squamous Cell Carcinoma

Objectives: Assessment of the cell proliferation rate in tissues can be one of the markers for impending malignancy in precancers. The state of activation and the proliferation activity of the cells can be assessed by the frequency of silver stained Nucleolar Organiser regions (AgNOR) within the nuclei which is significantly higher in malignant cells. The present study was carried out to analyze the distribution of the AgNOR in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC), and in their various histological grades, and to assess if the AgNOR distribution could give information on the malignant potentiality in premalignant lesions and aggressiveness of the malignant lesions. Study design: The study specimens comprised of 35 archival cases, of which 15 cases were of OL and 20 cases of OSCC. The specimens were stained by hematoxylin and eosin and modified silver staining method of Ploton et al. for the Nucleolar Organiser Regions. The specimens were analyzed independently by the two observers and was further statistically analysed. Results: The mean AgNOR count in OL was 2.80 ±0.50 and in cases of OSCC was 5.71± 1.08. The mean AgNOR count in OL cases of mild dysplasia was 2.59 ±0.66, in moderate dysplasia was 2.92± 0.43 and in severe dysplasia was 2.79. The mean AgNOR count in cases of well differentiated OSCC was 5.73± 1.62 and in cases of moderately differentiated OSCC was 5.67±1.19. Conclusion: The mean AgNOR count was higher in cases of OSCC as compared to cases of OL, and the AgNOR counts increased with the increase in the grades of dysplasia indicating a higher proliferative rate with increase in dysplasia.

Effects of Podoplanin on cell proliferation and cell cycle in oral leukoplakia cells

To investigate the effects of Podoplanin on cell proliferation and cell cycle in oral leukoplakia cells. Oral leukoplakia cell line (Leuk-1) transfected with pCMV-Podoplanin (A4-1) or pCMV (B4-1) was used in this study. The levels of mRNA and protein of Podoplanin were detected by real-time PCR and Western boltting in A4-1, B4-1 and Leuk-1 cells. The localization of Podoplanin was detected by confocal microscope. Cell growth and proliferation was examined by methyl thiazolyl tetrazolium (MTT) assay and cell cycle was detected by flow cytometry (FCM). The value of Podoplanin mRNA level in A4-1 cells was 0.022, which was significantly higher than the values in B4-1 (0.001) and Leuk-1 cells (0.002), P < 0.05. The gray scale of Podoplanin protein in A4-1 cells was significantly higher than in the control groups (P < 0.05). The expression of Podoplanin was observed in cell plasm and membrane of A4-1, B4-1 and Leuk-1 cells. But the expression level of Podoplanin in A4-1 cells was higher than in control groups. A4-1 cells grew faster than Leuk-1 cells. The proliferation rate after 3 days of culture in A4-1 cells was 40.4% higher than that in B4-1 cells (P < 0.05). The G₂-M phase (24.89 +/- 4.55)% and PI (0.57 +/- 0.06) of A4-1 cells were significantly higher than that in B4-1 cells [(4.13 +/- 5.24)%, (0.41 +/- 0.04)] and Leuk-1 cells [(4.69 +/- 7.42)%, (0.40 +/- 0.02)], P < 0.05. Over expression of Podoplanin accelerated the growth and proliferation of oral leukoplakia cells. Podoplanin may be one of key genes in the malignant transformation of oral leukoplakia.

In vitro and Clinical Studies of Gene Therapy with Recombinant Human Adenovirus-p53 Injection for Oral Leukoplakia

Oral leukoplakia is a well-recognized precancerous lesion of squamous cell carcinoma. When accompanied with abnormal p53 expression, it suffered a higher risk of canceration. The present study was carried out to test whether the recombinant human adenovirus-p53 could introduce wild-type p53 gene to oral leukoplakia cells and induce cell cycle arrest and apoptosis. We select p53(-) oral dysplastic keratinocyte POE-9n, to observe the growth inhibition, cell cycle change, apoptosis-induced effects, and elaborate the corresponding molecular mechanism of recombinant adenovirus-p53 on POE-9n cells. Meanwhile, we evaluate the feasibility, safety, and biological activity of multipoints intraepithelial injections of recombinant adenovirus-p53 in 22 patients with dysplastic oral leukoplakia. Exogenous p53 could be successfully transduced into POE-9n cells by recombinant adenovirus-p53. The optimal infecting titer in this study was multiplicity of infection (MOI) = 100. Recombinant adenovirus-p53 could strongly inhibit cell proliferation, induce apoptosis, and arrest cell cycle in stage G(1) in POE-9n cells by inducing p21(CIP/WAF) and downregulating bcl-2 expression. In the posttreatment patients, p53 protein and p21(CIP/WAF) protein expression were significantly enhanced, yet bcl-2 protein presented low expression. Sixteen patients showed clinical response to the treatment, and 14 patients showed obvious histopathologic improvement. Intraepithelial injections of recombinant human adenovirus-p53 were safe, feasible, and biologically active for patients with dysplastic oral leukoplakia.

High-Dose Fenretinide in Oral Leukoplakia

We previously showed that low-dose fenretinide (200 mg/d) had limited activity in retinoid-resistant oral leukoplakia (34% response rate) possibly because serum drug levels were insufficient to induce retinoid receptor-independent apoptosis. Therefore, we designed the single-arm phase II trial reported here to investigate whether higher-dose fenretinide would improve leukoplakia response over that of our previous study. Leukoplakia patients received fenretinide (900 mg/m(2) twice daily) in four 3-week cycles (1 week on drug followed by 2 weeks off). At week 12, clinical responses were determined and blood samples were collected for serum drug level assessments. A planned interim futility analysis led to early trial closure after the initial 15 (of 25 planned) patients because only 3 (20%) had a partial response (stopping rule: <or=4 responses in first 16 patients). Fenretinide was well tolerated--only one grade 3 adverse event (diarrhea) occurred. Serum fenretinide levels changed from 0 (baseline) to 0.122 +/- 0.093 micromol/L (week 12). In correlative in vitro studies, high-dose fenretinide inhibited the growth of head and neck cancer cells more and oral leukoplakia cells less than did lower doses of fenretinide. This result is consistent with our clinical finding that high-dose fenretinide did not improve on the historical response rate of lower-dose fenretinide in our previous oral leukoplakia trial.

Expression of p16 in oral cancer and premalignant lesions

Expression of p16 has been proposed as a marker for malignant transformation. This study aimed to evaluate p16 expression in oral squamous cell carcinoma (OSCC) and premalignant lesions including oral leukoplakia (OL) with and without dysplasia. Expression of p16 was investigated in 56 samples including OSCC, OL with and without dysplasia, and normal oral mucosa. Expression of p16 was identified by immunohistochemistry, using the CINtecTM p16INK4a Histology Kit. Both nuclear and/or cytoplasmic staining of the keratinocytes were considered to be positive and the percentage of positive cells was calculated. Expression of p16 was detected in 3/16 (18.75%) cases of OSCC, in 4/15 (26.7%) cases of OL without dysplasia, and in none of OL with dysplasia and normal mucosa. No significant differences in p16 expression prevalence were found among OSCC, OL with and without dysplasia and normal mucosa. The percentages of positive cells in OSCC and OL without dysplasia were 0.89 and 0.17, respectively. No significant difference in the percentage of positive keratinocytes was found. As a marker, p16 is not reliable for oral mucosal dysplasia and malignant transformation.

Genomics of Smoking Exposure and Cessation: Lessons for Cancer Prevention and Treatment

Tobacco use is the greatest preventable cause of cancer in the United States, accounting for almost one third of all cancerrelated deaths and 90% of deaths from lung cancer. Despite widespread knowledge of these risks, tobacco use prevalence rates are 20% in the United States and up to 30% to 50% in the developing world (1, 2). Nicotine, the addictive chemical in tobacco, produces a biological dependence (3), and therefore, even with the most efficacious medications available, only one in four smokers is able to maintain long-term abstinence (4). Persistent tobacco use is common even following a diagnosis of tobaccorelated cancer and is associated with poorer outcomes of radiation therapy and chemotherapy and with increased risk of second primary malignancies (5). Although smoking cessation is optimal, it may not be realistic for all patients. Therefore, it is essential to gain a better understanding of the cellular and molecular mechanisms through which tobacco exposure contributes to cancer pathogenesis and outcomes, and to develop targeted prevention and therapeutic approaches. Toward this end, a study reported by Gumus et al. (6) in this issue of the journal characterized gene expression profiles in the oral mucosa of smokers and nonsmokers and validated their findings by examining the in vitro effects of tobacco smoke condensate on gene expression in an oral leukoplakia cell line. Another related study also reported in this issue of the journal was conducted by Zhang et al. (7), who compared gene expression in cells obtained from bronchial brushes from never, current, and former smokers. Smoking effects on global gene expression were common across these studies. Certain genes (CYP1A1 and CYP1B1) that were up-regulated in buccal oral specimens of smokers were part of a larger group (CYP1A1, CYP1B1, ALDH3A1, NQO1, and AKR1C1) up-regulated following in vitro exposure to tobacco smoke condensate (6) and in the airway cells of smokers (7). Zhang and colleagues provide further in vivo evidence that many of these genes are down-regulated in persons who reported quitting smoking at least 1 year before the assessment (CYP1B1, AKR1C1, AKR1C2, AKR1B10 ,a ndALDH3A1). Many of these same genes were previously shown to be overexpressed in oral cancer cells exposed to tobacco smoke condensate (8), in bronchial epithelial cells of smokers, and in patients' non–small cell lung carcinoma cells (9, 10). The effects are also consistent with the effects of smoking on the airway transcriptome (11, 12). Taken together, these findings identify important tobacco smoke exposure response genes. The studies of Gumus et al. and Zhang et al. in this issue of the journal provide an elegant illustration of the value of cross-model (preclinical and clinical) validation in translational cancer prevention research.

Mycoplasma infection in the epithelial cells of oral leukoplakia

Mycoplasma infection in the epithelial cells of oral leukoplakia

Invasion of mycoplasma in epithelial cells of oral leukoplakia

Invasion of mycoplasma in epithelial cells of oral leukoplakia

A quantitative assessment of Langerhans cells in oral mucosal lichen planus and leukoplakia.

Quantitative analysis of Langerhans cells stained with OKT6 in the epithelium of 42 patients with oral mucosal lichen planus and 14 patients with oral mucosal leukoplakia showed significantly greater numbers of Langerhans cells in lichen planus. This may reflect the role of Langerhans cells in the pathogenesis of this condition and their identification could help in distinguishing lichen planus from leukoplakia.

Epstein–Barr virus in oral diseases

Epstein-Barr virus (EBV), a B-lymphotropic gamma-herpesvirus, causes infectious mononucleosis and oral hairy leukoplakia, and is associated with various types of lymphoid and epithelial malignancies. Saliva is the main vehicle for EBV transmission from individual to individual. Recent studies have also implicated EBV in the pathogenesis of advanced types of periodontal disease. EBV DNA is detected in 60-80% of aggressive periodontitis lesions and in 15-20% of gingivitis lesions or normal periodontal sites. The periodontal presence of EBV is associated with an elevated occurrence of periodontopathic anaerobic bacteria. Moreover, EBV active infection occurs in approximately 70% of symptomatic and large-size periapical lesions. EBV and cytomegalovirus often co-exist in marginal and apical periodontitis. Periodontal therapy can markedly suppress the EBV load in periodontal pockets as well as in saliva, which has the potential to reduce the risk of viral transmission between close individuals. EBV proteins up-regulate cytokines and growth factors, which seem to play a central role in the proliferative response of tongue epithelial cells in oral hairy leukoplakia and in the cell-transformation process of EBV-associated malignancies. Further research is needed to identify the full range of EBV-related diseases in the human oral cavity.

Histone Deacetylase Inhibitors Suppress the Induction of c-Jun and Its Target Genes Including COX-2

Cyclooxygenase-2 (COX-2) is considered to be a target for anticancer therapy. Histone deacetylase (HDAC) inhibitors exhibit antitumor activity, but the mechanisms of action are incompletely understood. We investigated whether HDAC inhibitors blocked AP-1-mediated activation of COX-2 transcription. Trichostatin A and suberoylanilide hydroxamic acid, two structurally related inhibitors of HDAC activity, blocked AP-1-mediated induction of COX-2 expression and prostaglandin E2 biosynthesis. Chromatin immunoprecipitation assays indicated that HDAC inhibitors suppressed c-Jun binding to the COX-2 promoter and thereby blocked transcription. The observed reduction in binding reflected reduced levels of c-Jun. HDAC inhibitors suppressed the induction of c-jun transcription by blocking the recruitment of the preinitiation complex (RNA polymerase II and TFIIB) to the c-jun promoter. HDAC3 but not HDAC1 or HDAC2 was required for AP-1-mediated stimulation of c-jun expression. Because HDAC inhibitors suppressed the induction of c-jun gene expression, resulting in reduced COX-2 transcription, it was important to determine whether other known AP-1 target genes were also modulated. Cyclin D1 and collagenase-1 are AP-1-dependent genes that have been implicated in carcinogenesis. HDAC inhibitors suppressed the induction of both cyclin D1 and collagenase-1 transcription by inhibiting the binding of c-Jun to the respective promoters. Taken together, these results suggest that HDAC inhibitors block the induction of c-jun transcription by inhibiting the recruitment of the preinitiation complex to the c-jun promoter. This led, in turn, to reduced expression of several activator protein-1-dependent genes (COX-2, cyclin D1, collagenase-1). These findings provide new insights into the mechanisms underlying the antitumor activity of HDAC inhibitors.

Retracted: The evolution of predictive oncology and molecular‐based therapy for oral cancer prevention

More than 300,000 new cases worldwide are being diagnosed with oral squamous cell carcinoma annually. This aggressive epithelial malignancy is associated with a high mortality and severe morbidity among the long-term survivors. The ability to intervene prior to this advanced stage may improve treatment results. This requires the early identification of molecular events that represent early phases of malignant transition, which is possible through measurement of DNA ploidy in epithelial cells of oral leukoplakia. Recently, we showed that patients with aneuploid dysplastic oral lesions had a 96% rate of oral cancer (26 of the 27 patients received the diagnosis) with a 70% rate within three years, an 81% rate of subsequent cancer (22 of 27), a 74% rate of death from cancer (21 of 27) and virtually no help from complete resection-all hallmarks of biologically aggressive cancer. Standard treatment of oral leukoplakia-a precursor lesion of oral cancer-varies from watchful waiting to complete resection. We have recently demonstrated that complete resection of aneuploid oral leukoplakia does not prevent the occurrence of clinically aggressive and highly lethal oral cancer. Oral carcinogenesis is a complex multifocal process of multiclonal field carcinogenesis and intraepithelial clonal spread. The multifocal nature of early oral carcinogenesis may hinder local treatment modalities. Inhibitors of cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR), either alone or in combination, may be used for reversing or stopping the oral carcinogenesis at an early stage of disease. The failure of standard treatment to control aneuploid oral leukoplakia justifies clinical trials with new treatment modalities, such as systemic therapy with molecular targeting agents, which in patients with aggressive leukoplakia is tantamount to cancer therapy.

The p16 methylation in oral leukoplakia and oral squamous cell carcinoma

To investigate p16 gene methylation in normal mucosa, leukoplakia with hyperplasia and dysplasia and oral squamous cell carcinoma. 20 patients of leukoplakia with hyperplasia, 11 patients of leukoplakia with mild dysplasia, 10 patients of leukoplakia with moderate dysplasia, 9 patients of leukoplakia with severe dysplasia, 10 patients with OSCC in low grade, 12 patients with OSCC in moderate grade, 8 patients with OSCC in high grade, and 10 normal individuals were studied on p16 methylation. Rates of p16 methylation were 0 for normal individuals, 5% for patients of leukoplakia with hyperplasia, 18% for patients of leukoplakia with mild dysplasia, 10% for patients of leukoplakia with moderate dysplasia, 22% for patients of leukoplakia with moderate dysplasia, and 50% for patients with OSCC in low grade, 42% for patients with OSCC in moderate grade, and 63% for patients with OSCC in high grade. Rates of p16 methylation increased with tissue malignance increase and correlated positively to the tissue malignance. The rate (82%) of p16 methylation in OSCC patients with lymph node metastasis was significantly high than that (32%) of OSCC patients without lymph nodes metastasis. The methylated rates of p16 correlated positively to the lacking rates of p16 protein. It was first reported that p16 methylation occurred in every stage of leukoplakia cancerization and OSCC progression. p16 can be one of the important molecular biological markers for leukoplakia cancerization and OSCC progression. p16 methylation is recommended as an important marker for diagnosis of OSCC.

Intracellular localization of the Epstein-Barr virus BFRF1 gene product in lymphoid cell lines and oral hairy leukoplakia lesions.

A novel protein encoded by the BFRF1 gene of the Epstein-Barr virus was identified recently [Farina et al. (2000) J Virol 74:3235-3244], which is antigenic "in vivo" and expressed early in the viral replicative cycle. In the present study, its subcellular localization was examined in greater detail comparing Epstein-Barr virus (EBV) induced producing and nonproducing cell lines by immunofluorescence: in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-induced Raji and B95-8 cells, as well as in anti-IgG-stimulated Akata cells, the protein appeared to be localized over the cell nuclear membrane. A similar nuclear membrane localization was observed in epithelial cells of oral hairy leukoplakia, a pathological manifestation of permissive EBV infection. In contrast, upon transfection of BFRF1 in the EBV-negative Burkitt's lymphoma cell line DG75, the protein was localized predominantly over the plasma membrane. The membrane localization was abolished when DG75 cells were transfected with a C-terminal deletion mutant of BFRF1 lacking the transmembrane domain. Because induced Raji cells do not produce virus, the above observations indicate that the nuclear membrane localization is not associated with viral production, but requires the expression of EBV genes, and suggest that additional proteins, expressed early during viral lytic infection, might be necessary to target the protein to the nuclear membrane. Immunogold electron microscopy on ultrathin cryosections of induced B95-8 cells showed that BFRF1 on the nuclear membranes was concentrated over multilayered domains representing areas of active viral replication or at the sites of viral budding, suggesting that BFRF1 is involved in the process of viral assembly.

Evaluation of TGF-α and EGFR Expression in Oral Leukoplakia and Oral Submucous Fibrosis by Quantitative Immunohistochemistry

Objective: Oral leukoplakia (OL) and oral submucous fibrosis (OSMF) are clinically distinct preneoplastic states that precede the development of oral squamous cell carcinoma. Recently, attempts are being made to identify specific molecular event(s) as prognostic markers to identify oral precancerous lesions with higher malignant potential. The goal of the present study was to evaluate the expression of EGFR and its ligand TGF-α in OL with dysplasia and OSMF as intermediate markers of malignancy by quantitative immunohistochemistry. Methods: Oral tissues were stained with monoclonal antibodies to TGF-α and EGFR. The results were analyzed with Photoquant image analysis software. Results: The expression of TGF-α and EGFR was upregulated in OL, OSMF and oral squamous cell carcinomas relative to normal oral mucosa. The area and intensity of staining of TGF-α in the proliferative layers (stratum germinativum) increased linearly in OL with mild, moderate and severe dysplasia as compared to control mucosa (p < 0.05). EGFR levels increased linearly in the stratum spinosum in OL with increasing degrees of dysplasia (p < 0.05). In general, the expression of both proteins in OSMF was similar to that in OL with moderate dysplasia. Conclusions: EGFR and TGF-α represent early markers of malignancy in OL with dysplasia. Quantitative measurement of these proteins may provide intermediate endpoints in prospective chemopreventive trials.

In situ staining with DNA-binding fluorescent dye, Hoechst 33258, to detect microorganisms in the epithelial cells of oral leukoplakia.

This study was performed to investigate the presence of microorganisms in the epithelial cells of leukoplakia. Frozen sections of 20 specimens of leukoplakia were stained with DNA-binding bisbenzimide Hoechst 33258. As a control, 20 specimens of normal oral mucosa and five specimens of normal skin were used. In all preparations of leukoplakia, small granular fluorescing structures were observed within the cytoplasm of the epithelial cells, predominantly within the cytoplasm of prickle cells, although the amount of the granular structures varied between specimens, layers of the epithelium and even areas of the epithelium within a single section. Less granular structures were observed, or none at all, in the cytoplasm of the epithelial cells of normal mucosa. No structures were observed in the cytoplasm of the epithelium of skin. The results in this study strongly suggest that microorganisms are present in the epithelial cells of oral mucosa, and that they are closely associated with the development of oral leukoplakia. It is postulated that the microorganisms in the epithelial cells could be bacteria, particularly mycoplasmas.