Osteopontin (OPN) distribution in premalignant and malignant lesions of oral epithelium and expression in cell lines derived from squamous cell carcinoma of the oral cavity.

The objectives of this study were to assess the immunolocalization of human osteopontin (OPN) in oral lesions and to identify human cell lines of oral squamous cell carcinoma (OSCC) origin that express OPN mRNA. OPN was localized using immunohistochemistry in the following oral specimens: normal epithelium (n=6), epithelial hyperplasia (n=4), epithelial dysplasia (n=28), carcinoma in situ (n=11) and squamous cell carcinoma (n=43). Cell lines UMSCC-1, MDA TU 138, MDA 686LN, SCC4, SCC9, SCC25, CAL 27 and MDA 1483 were characterized for OPN mRNA expression using Northern blotting. OPN was not detected in normal oral epithelium. Intracellular and intercellular immunoreactivity was seen in 75% of hyperplasias, 57% of dysplasias, 54% of carcinoma in situ and 67% of squamous cell carcinomas. UMSCC-1 expressed high levels of OPN mRNA. We conclude that OPN protein is detectable in premalignant and malignant lesions arising from oral epithelium. UMSCC-1 may be a useful cell line in which to conduct in vitro studies designed to clarify the role of OPN in OSCC.

Variable expression of cytokines in human head and neck squamous cell carcinoma cell lines and consistent expression in surgical specimens.

Head and neck squamous cell carcinomas (HNSCCs) are associated with abnormal cell-mediated immunity at the primary tumor site. We investigated tumor-derived cytokines as factors underlying such abnormalities. Cytokine mRNA and protein of eight HNSCC-derived cell lines were tested; reverse transcription-PCR results indicated the presence of mRNAs for interleukin 1alpha (IL-1alpha) and transforming growth factor alpha (8 of 8); transforming growth factor beta and IL-1beta (7 of 8); and IL-4 and IL-6 (4 of 8). IL-2, IFN-gamma, and tumor necrosis factor alpha mRNA were not detected. Supernatants from six of these cell lines were analyzed by ELISA; IL-1alpha, IL-1beta, and IL-6 were found to be markedly increased compared to human papillomavirus-16-immortalized human oral keratinocytes. To determine whether the cell line findings are applicable to primary tumors, we performed immunohistochemical analysis on tumor specimens from 12 patients with invasive HNSCC. Universal intracellular production of IL-1alpha, IL-1beta, and IL-6 protein was detected. We conclude that the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients.

Interleukin-1 regulates interleukin-6 secretion in human oral squamous cell carcinoma in vitro: possible influence of p53 but not human papillomavirus E6/E7.

We have previously shown that interleukin-1 (IL-1) and IL-6 are constitutively produced by human oral squamous cell carcinoma (SCC) and some derived cell lines but not by cultured normal oral keratinocytes. To elucidate possible cytokine regulatory pathways that may contribute to oral SCC growth and/or progression, we tested the hypotheses that exogenous and/or endogenous IL-1 regulates IL-6 production in vitro. We investigated the effects of exogenous IL-1 and IL-6 on secondary cytokine secretion. Our studies revealed that IL-1 strongly up-regulated IL-6 protein secretion in all three cell lines tested. This effect was completely abrogated by IL-1 receptor antagonist. IL-1 receptor antagonist also inhibited the secretion of IL-1alpha and IL-1beta in two of three cell lines. These data show for the first time that IL-1 strongly up-regulates IL-6 and support the notion of autocrine regulation of IL-1 in certain oral SCC cell lines. Additionally, because human papillomavirus (HPV) infection and p53 mutation have been implicated in the malignant transformation of SCC, we explored a second hypothesis, that HPV and/or p53 mutation contribute to cytokine dis-regulation. We investigated HPV DNA presence, transcriptional activation of HPV E6/E7 (in HPV DNA-positive cell lines), and p53 gene status in our cell lines. No association between HPV DNA and cytokine expression was found. However, the oral SCC cell lines secreting the most IL-6 had mutant rather than wild-type p53.

Evaluation of tyrosinase mRNA as a tumor marker in the blood of melanoma patients.

PURPOSE: The value of tyrosinase messenger RNA (mRNA) detection in the peripheral blood by reverse-transcription polymerase chain reaction (RT-PCR) as a melanoma marker remains controversial. The purpose of this study was to compare the sensitivities of two different blood processing techniques for tyrosinase mRNA detection and evaluate its potential clinical value. METHODS: A total of 50 patients with progressive stage IV melanoma was studied. Two blood processing methods were used: RNA extraction from the whole blood and RNA extraction from density gradient-isolated peripheral-blood mononuclear cells (PBMC). The RNA samples were tested with a sensitive nested-primer RT-PCR assay. RT-PCR results were also correlated with serum lactate dehydrogenase (LDH), treatment status, and presence of visceral versus nonvisceral metastases. RESULTS: Thirteen (26%) of the density gradient and five (10%) of the whole blood processed samples were PCR positive (P = .011). Serum LDH levels were found to be significantly higher in PCR-positive PBMC-processed patients (P = .015). There was no significant difference in the detection rates between visceral versus nonvisceral metastases or between prior treatment versus no prior treatment. CONCLUSION: Using a density gradient method to process the blood samples resulted in a higher detection rate of tyrosinase mRNA than extracting the RNA from the whole blood. However, the relatively low sensitivity in patients with disseminated and progressive disease compared with other reports suggests that tyrosinase mRNA may be of limited value in the management of malignant melanoma.

Interleukin-6 production and secretion in human melanoma cell lines: regulation by interleukin-1.

These investigations were designed to test the hypothesis that exogenous and/or endogenous interleukin-1 (IL-1) regulates interleukin-6 (IL-6) production in human melanoma cell lines. Ten cell lines were examined for IL-1 and IL-6 expression. Six of these 10 lines constitutively expressed detectable IL-6 mRNA by RT-PCR; three of these six cell lines also produced intracellular and secreted IL-6 as evidenced by positive reaction for IL-6 using immunohistochemistry staining and ELISA methods; three others produced only intracellular IL-6. Addition of exogenous IL-1 alpha was shown to have the following effects on IL-6 production: first, de novo induction of detectable IL-6 intracellular protein and secreted IL-6 in a cell line void of either; second, stimulation of IL-6 secretion in all three cell lines producing only intracellular IL-6 protein; and third, quantitative enhancement of IL-6 secretion in cell lines that constitutively secreted IL-6. Three of the 10 lines which secreted IL-6 also constitutively secreted IL-1 alpha. Experiments employing the IL-1 receptor antagonist confirmed an extracellular receptor-mediated role of IL-1 in regulation of IL-6 production in such cells. These results indicate that IL-1 can regulate IL-6 in human melanoma cells; however, heterogeneity in the constitutive expression as well as variability in response exists with respect to these two cytokines.

Analysis of human papillomavirus DNA in oral squamous cell carcinomas.

Evidence from several laboratories suggests that HPV plays a role in the etiology of squamous cell carcinomas of the oral cavity. A multifactorial risk factor profile for the development of oral cancer may include HPV in addition to well-established risk factors such as tobacco and alcohol use. The prevalence of oral carcinomas reported to be associated with HPV has varied widely due to differences in the sensitivity of the assay used for HPV detection. The aims of this study were: (1) to ascertain the prevalence of HPV DNA in oral squamous cell carcinomas using the most sensitive technique available, the polymerase chain reaction; (2) to determine the type of HPV in the tumors; and 3) to correlate the virologic data with other risk factor data obtained from patients' records. Fourteen (78%) of 18 primary tumors, 6 (67%) of 9 normal epithelial tissues from the patients and 5 (100%) of 5 neck metastases were HPV DNA-positive. Of the 14 HPV DNA-positive primary tumors, specific typing revealed HPV 16 in 2, HPV 18 in 2, HPV 16 and 18 in 5, HPV 6/11, 16 and 18 in 4, and HPV 6/11 in 1. HPV types in the normal or metastatic tissue were usually the same as those in the respective primary tumor. There was no significant association between HPV presence and any of 12 factors or patient characteristics studied.

Prevalence of Epstein-Barr virus and human papillomavirus in oral mucosa of HIV-infected patients.

Epstein-Barr virus (EBV) has been implicated in the genesis of oral hairy leukoplakia (OHL). Initially, OHL was also associated with human papillomavirus (HPV) as evidenced by staining with antiserum to papillomavirus common structural antigens and reports of two HPV-positive OHL as detected by in situ DNA hybridization. The aims of this study were to determine the prevalence of EBV and HPV DNA in OHL and normal oral mucosa and to explain the basis for the staining of OHL tissues with antibodies to papillomavirus common structural antigens. EBV DNA was detected by in situ hybridization in 47 of 47 cases of OHL from human immunodeficiency virus (HIV)-seropositive individuals and in 1 of 10 biopsies of clinically normal buccal mucosa from the same group of individuals. Twenty-five of 35 OHL specimens stained with antibody to papillomavirus common structural antigens. There was no staining of two EBV-containing lymphoblastoid lines, indicating that the staining with anti-papillomavirus antibody was not due to antigenic cross-reactivity with EBV antigens. HPV DNA was detected by polymerase chain reaction amplification in 10 of 18 OHL specimens and in 6 of 10 normal buccal mucosa specimens. Our results indicate that EBV and HPV are present frequently in OHL and that HPV can be found regularly in histologically normal mucosa.

Human papillomavirus DNA in tissue biopsy specimens of vulvar vestibulitis patients treated with interferon.

Thirteen women with the diagnosis of vulvar vestibulitis based on clinical symptoms, presence of vestibular tenderness on physical examination, and acetowhite changes of the vulvar vestibule were treated with intradermal injection of alpha-interferon. Biopsies of the acetowhite areas were analyzed for human papillomavirus (HPV) DNA using polymerase chain reaction amplification and dot blot hybridization. Eleven of 13 subjects harbored one of the HPV DNA types; six of these were type 16 and/or 18 and the others were unidentified. Five subjects (all HPV DNA-positive) reported resolution of symptoms with interferon therapy. Our results indicate the presence of HPV DNA in a subset of patients with vulvar vestibulitis, but its presence is not predictive of response to interferon therapy.

Cervical carcinoma metastatic to the skull, heart, and lungs: analysis for human papillomavirus DNA.

In this case report we present a 21-year-old patient with stage IV cervical carcinoma metastatic to the skull at the time of diagnosis. The patient had two previous negative Pap smears performed 10 months and 15 months prior to the time of diagnosis. Postmortem examination 2 months following diagnosis showed cardiac and pulmonary metastases. Human papillomavirus (HPV) DNA was detected in the primary cervical tumor and skull metastasis, but absent in the cardiac and pulmonary metastases. This study details the first report in the medical literature of a skull metastasis present at the time of diagnosis of cervical carcinoma. It also represents the first report of HPV-negative metastases arising from an HPV-positive cervical cancer.