Leukoplakia Cell Research Articles

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.

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.

P225 Expression of sexual hormone receptors in leukoplakia and oral squamous cell carcinoma: a case control study

P225 Expression of sexual hormone receptors in leukoplakia and oral squamous cell carcinoma: a case control study

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.

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.

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.

Cytomorphometric analysis of squames obtained from normal oral mucosa and lesions of oral leukoplakia and squamous cell carcinoma

Cytomorphometric analysis of squames obtained from normal oral mucosa and lesions of oral leukoplakia and squamous cell carcinoma

THE ESTIMATION OF PROLIFERATIVE ACTIVITY BY PCNA AND AGNORS IN LEUKOPLAKIA AND SQUAMOUS-CELL CARCINOMA OF THE ORAL CAVITY

The proliferating activity of leukoplakia and squamous cell carcinoma (SCC) was estimated using proliferating cell nuclear antigen (PCNA) immunostaining and silver-binding argyrophilic nucleolar organizer region (AgNOR) staining. Twenty-eight leukoplakias and 15 SCCs of the oral cavity were used in this study. The mean+/-S.D. of PCNA LI and AgNOR counts were 7.7+/-6.0% and 2.43+/-0.68/nucleus in leukoplakias, and 22.3+/-11.6% and 4.77+/-1.49/nucleus in SCCs. Both of PCNA LI and AgNOR counts were significantly higher in SCCs than in leukoplakias. There was a significant linear correlation between PCNA LI and AgNOR counts (p=0.0022) in leukoplakias, but not in SCCs. In the series of leukoplakias, PCNA LI apparently increased in leukoplakias with dysplasia and malignant transformed cases. Our data suggest that PCNA LI and AgNOR counts are useful markers of proliferating activity, and PCNA LI might be a prognostic factor of leukoplakias.

Epstein-Barr virus in the proliferative diseases of squamous epithelium in the oral cavity

The presence of Epstein-Barr virus was analyzed in 79 cases of oral epithelial proliferative diseases by polymerase chain reaction, in situ hybridization for Epstein-Barr virus-deoxyribonucleic acid and Epstein-Barr virus-encoded small messenger ribonucleic acid and immunohistochemistry for Epstein-Barr virus latent membrane protein. These lesions were histologically categorized as invasive squamous cell carcinoma (n = 36), carcinoma in situ (n = 10), verrucous carcinoma (n = 4), leukoplakia (n = 19), and papilloma (n = 10). Epstein-Barr virus genomes were detected in 19 squamous cell carcinoma (52.8%), four carcinoma in situ (40%), and one leukoplakia (5.3%); none of the verrucous carcinoma or papilloma cases were positive with polymerase chain reaction. By deoxyribonucleic acid in situ hybridization, positive signals were observed in the nuclei of cancer cells in 10 cases, in infiltrating lymphocytes in three, and both in one case. In patients with carcinoma in situ, only a single case was positive. In one case of leukoplakia positive signals were found in upper and middle layer squamous cells. The results by Epstein-Barr virus-encoded small messenger ribonucleic acid in situ hybridization revealed the same distribution as that by deoxyribonucleic acid in situ hybridization. Latent membrane protein was expressed only in the epithelial cells of leukoplakia but not in cases with squamous cell carcinoma and carcinoma in situ. These findings suggest that Epstein-Barr virus infection of oral squamous epithelium may be carcinogenic; alternatively, the virus may merely exist in epithelial cells of squamous cell carcinoma, carcinoma in situ, and leukoplakia as a passenger.

Tissue binding patterns of eight different lectins in normal, leukoplakic and malignant lesions of the oral cavity

The present investigation was undertaken to assess the binding characteristics of eight different lectins on normal mucosa (11 cases), Leukoplakia (5 cases) and squamous cell carcinoma (12 cases) by the use of biotinylated lectins and avidin biotin peroxidase complex. The lectins employed were soybean (SBA), peanut (PNA) Dolichos biflorus (DBA) Concanavalin A (CON A), Wheat germ (WGA), ulex Europeus(UEA), Ricinus communic (RCA) and Lotus tetragonolobus (LTA). It was noticed that SBA, DBA, WGA, UEA, RCA and LTA showed intense to high binding in normal oral mucosa but absent or very weak binding in squamous cell carcinoma. PNA on the contrary had a weak binding to normal mucosal epithelial cells but strong binding to malignant cells. The Leukoplakic mucosa had an intermediate binding pattern. The Lectin CON A was not bound at all or seen in very low concentration in the malignant cells and leukoplakic epithelium but showed weak bindng in the normal mucosa. Hence it appears that Lectin binding studies may provide information about functional differences between cells as the result of normal maturation or pathological changes.

Immunohistochemical localization of epidermal growth factor (EGF) and EGF receptor in human oral mucosa and its malignancy

The immunohistochemical localizations of human epidermal growth factor (hEGF) and EGF receptor (EGFr) in oral tissues, including normal mucosa, leukoplakia and squamous cell carcinoma were examined by the use of monoclonal antibodies to hEGF and EGFr. In normal mucosa and leukoplakia, immunostaining of hEGF was limited to an underlying layer of connective tissue near the epithelium. The intensity of extracellular staining appeared to increase with the degree of epithelial malignancy and was eventually most striking in the stroma of invasive carcinoma. The epithelial cells in normal mucosa, leukoplakia, and squamous cell carcinoma showed negligible immunoreactivity for hEGF. Expression of EGFr appeared to be associated with the proliferative activity of cells and/or epithelial malignancy. In normal mucosa, anti-EGFr monoclonal antibody reacted only with the basal cell layer. In all sections of leukoplakia, the positive cells for EGFr were found in the prickle cell layer in addition to the basal cell layer. Most tumour cells in squamous cell carcinoma were strongly positive for EGFr. These findings indicate increased expression of hEGF and EGFr with malignancy. The characteristic localization of extracellular hEGF in the underlying connective tissue and in stroma of oral mucosal tumours suggests a possible epithelial-mesenchymal interaction in hEGF secretion.

Inhibition of a cell surface protease after cisplatin chemotherapy.

The epithelial cells in squamous carcinoma and leukoplakia of the oral cavity possess the cell surface protease, guanidinobenzoatase (GB), in an active form. GB is closely similar to plasminogen activator, a protease associated with both transformed cells and tumour cells. The active centre of GB binds the fluorescent probe 9-aminoacridine (9-AA) enabling cells containing active GB to be visualised by fluorescent microscopy. It was observed that chemotherapy with cisplatin resulted in a marked decrease in cell surface GB activity and this decrease was due to the formation of an enzyme-inhibitor complex. One of the results of chemotherapy was shown to be the suppression of a cell surface protease which is known to be associated with migration and malignancy of cells in vivo.

Epstein-Barr virus small nuclear RNAs are not expressed in permissively infected cells in AIDS-associated leukoplakia.

Epstein-Barr virus (EBV) DNA structure and gene expression were analyzed in tissue specimens from oral hairy leukoplakia (HLP), a mucocutaneous lesion that develops in patients infected with human immunodeficiency virus (HIV). The structure of the terminal restriction enzyme fragments of EBV revealed that HLP is a permissive infection without a predominant, detectable population of EBV episomal DNA. In RNA preparations from this uniquely permissive infection, EBV replicative mRNAs could be identified by Northern analysis; however, the virally encoded small nuclear RNAs, the EBERs, were not detected in most HLP RNA preparations. In situ hybridization detected EBER expression in very rare cells. These data indicate that unlike other viral small nuclear RNAs, the EBERs are not expressed during viral replication and must participate in the complex maintenance of latent EBV infection.

Epstein-Barr virus small nuclear RNAs are not expressed in permissively infected cells in AIDS-associated leukoplakia.

Epstein-Barr virus small nuclear RNAs are not expressed in permissively infected cells in AIDS-associated leukoplakia.

Further ultrastructural findings in epithelial cells of hairy leukoplakia

Epstein-Barr virus and multivesicular structures appearing in lesions of hairy leukoplakia have been examined ultrastructurally in serial sections. It was found that both structures probably represent various levels of sectioning of transversely cut cell processes. The so-called viral nucleocapsid having a single membrane results when the plane of section is at right angles to one of the membranes but tangential to the other. While the so-called viral particle having two membranes would result if the plane of section is perpendicular to both the membranes of adjacent cells. The internal electron-dense core probably represents filaments enclosed within these processes and therefore does not represent DNA nucleotides. In addition, these nuclei contained bundles of filaments which probably represented tonofilaments. This was found to be due to tangential sectioning of the nuclear membrane surrounding deep cytoplasmic invaginations which led to the appearance of these filaments as if free within the nucleus.

Replication of Epstein-Barr virus within the epithelial cells of oral "hairy" leukoplakia, an AIDS-associated lesion.

We conducted a study to identify the viruses in tissue specimens of oral "hairy" leukoplakia, a lesion that is found in immunosuppressed male homosexuals and that is associated with the subsequent development of the acquired immunodeficiency syndrome. When stained for papillomavirus core antigen, 49 of 67 biopsy specimens (73 per cent) yielded positive results in epithelial-cell nuclei. Electron microscopy showed papillomavirus-like particles in all of 25 specimens, and the herpes-type virus described in a previous report was seen in 23 of the 25 specimens. Three specimens had both types of particle in the same individual epithelial cells. Immunofluorescence for herpes simplex virus, varicella-zoster virus, and cytomegalovirus gave negative results in all cases, but 19 of 20 specimens showed intense nuclear staining in epithelial cells for the viral capsid antigen of Epstein-Barr virus (EBV). DNA hybridization using EBV probes in Southern blots demonstrated EBV DNA in all of 13 specimens and found 200 or more viral DNA molecules per cellular genome in 11 of the 13. The whole EBV genome was also demonstrated in the specimens and found to be in linear virion form. We conclude that EBV replicates within the epithelial cells in hairy leukoplakia.

Oral viral leukoplakia--a new AIDS-associated condition.

Viral leukoplakia of the tongue in immunosuppressed male homosexuals (oral “hairy” leukoplakia, H.L.) is a newly described lesion showing an association with the subsequent development of AIDS, often with Pneumocystis carinii pneumonia. The lesion appears to contain papillomavirus as well as a second virus of the herpes-group (1). In this paper we present evidence for the presence, within the epithelial cells of hairy leukoplakia, of both papillomavirus and Epstein-Barr virus (EBV), and for the presence of EB viral capsid antigen (VCA).

Study of biopsies from lesions of the oral mucosa by scanning electron microscopy (S.E.M.)

Surface epithelial cells from 55 biopsies including lichen planus, leukoplakia and squamous cell carcinoma were studied by scanning electron microscopy. The surface structures of the various oral lesions demonstrated morphological differences. After more information has been collected, the method of SEM may be of value for the recognition of pre- and early neoplastic lesions in the oral cavity.

Investigation of DNA-content of leukoplakia cells of the oral mucosa

Investigation of DNA-content of leukoplakia cells of the oral mucosa

Hypopharyngeal and Laryngeal Involvement With Darier Disease

The presence of mucosal Darier disease (keratosis follicularis) was evaluated clinically and histologically in four consecutive patients with moderate to extensive cutaneous manifestations of this disease. Fiberoptic endoscopy demonstrated hypopharyngeal or laryngeal involvement or both in each patient. A dearth of corps ronds and grains in these anatomical regions was observed histologically. The routine evaluation of mucosal involvement in Darier disease should result in a higher incidence of hypopharyngeal and laryngeal lesions than is currently known. Furthermore, the recognition of hypopharyngeal and laryngeal involvement with Darier disease must be emphasized because of the clinical similarity to leukoplakia and squamous cell carcinoma.