Comparison of the miRNA profiles in HPV-positive and HPV-negative tonsillar tumors and a model system of human keratinocyte clones.

Background: Better insights into the molecular changes involved in virus-associated and -independent head and neck cancer may advance our knowledge of HNC carcinogenesis and identify critical disease biomarkers. Here we aimed to characterize the expression profiles in a matched set of well-characterized HPV-dependent and HPV-independent tonsillar tumors and equivalent immortalized keratinocyte clones to define potential and clinically relevant biomarkers of HNC of different etiology.

Methods: Fresh frozen tonsillar cancer tissues were analyzed together with non-malignant tonsillar tissues and compared with cervical tumors and normal cervical tissues.

Analysis of the integration of human papillomaviruses in head and neck tumours in relation to patients' prognosis.

Integration, which leads to the disruption of the circular HPV genome, is considered as a critical, albeit not obligatory, step in carcinogenic progression. Although cervical carcinomas with extrachromosomal HPV plasmid genomes have been described, the virus is integrated in 70% of HPV16-positive cervical tumours. Limited information is available about HPV integration in head and neck tumours (HNC).

Interferon treatment of human keratinocytes harboring extrachromosomal, persistent HPV-16 plasmid genomes induces de novo viral integration.

Interferons (IFNs) have been used to treat epithelial lesions caused by human papillomavirus (HPV) persistence. Here, we exposed primary human keratinocytes (HFKs) immortalized by persistently replicating HPV-16 plasmid genomes to increasing levels of IFN-γ. While untreated HFKs retained replicating HPV-16 plasmids for up to 60-120 population doublings, IFN led to rapid HPV-16 plasmid loss.

Analyzing the Human Papillomavirus (HPV) Life Cycle in Primary Keratinocytes with a Quantitative Colony-Forming Assay.

Papillomavirus genomes replicate as extrachromosomal plasmids within infected keratinocytes, requiring the regulated expression of early viral gene products to initially amplify the viral genomes and subvert cell growth checkpoints as part of a complex path to immortalization. Building on contemporary keratinocyte transfection and culture systems, the methods described in this unit form a detailed approach to analyzing critical events in the human papillomavirus (HPV) life cycle, utilizing physiologic levels of viral gene products expressed from their native promoter(s) in the natural host cells for HPV infection. A quantitative colony-forming assay permits comparison of the capacities of various transfected HPV types and mutant HPV genomes to initially form colonies and immortalize human keratinocytes.

The truncated C-terminal E2 (E2-TR) protein of bovine papillomavirus (BPV) type-1 is a transactivator that modulates transcription in vivo and in vitro in a manner distinct from the E2-TA and E8^E2 gene products.

The E2 open reading frame of bovine papillomavirus (BPV)-1 encodes a 410 amino acid (aa) transcriptional activator, E2-TA, and collinear polypeptides--E2-TR (243 aa) and E8^E2 (196 aa). E8^E2 and E2-TR share the DNA-binding domain of E2-TA, and both have been defined as transcriptional repressors. Although purified E2-TR and E8^E2 proteins specifically bound E2 sites with similar affinities, only the E2-TR stimulated transcription.

Complex etiology underlies risk and survival in head and neck cancer human papillomavirus, tobacco, and alcohol: a case for multifactor disease.

Findings are inconsistent about whether tobacco, alcohol, and human papillomavirus (HPV) are two independent HNC risk factor groups that distinguish an infection-associated cancer from a tobacco/alcohol-associated HNC. We found that cancer in the oral cavity risk was greater in HPV-E6/E7 seropositive/heavy tobacco users (adjusted OR = 3.5) than in HPV-seronegative/heavy tobacco users (adjusted OR = 1.

Human papillomavirus serologic follow-up response and relationship to survival in head and neck cancer: a case-comparison study.

Background: Human papillomavirus high risk (HPV-HR) type 16 is a significant risk factor for head and neck cancers (HNC) independent of tobacco and alcohol. The purpose of this study was to determine whether antibody levels to the HPV-16 oncoproteins E6 and E7 measured in sera collected at baseline (BL) prior to treatment and at two post-treatment follow-up (FU) visits were associated with HNC risk factors or prognosis.

Methods: Presence of antibodies to HPV-16 E6 and E7 was evaluated in 109 newly diagnosed HNC cases with BL and FU blood samples, using the enzyme-linked immunosorbent assay (ELISA).

Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers.

Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal.

Tobacco and alcohol use increases the risk of both HPV-associated and HPV-independent head and neck cancers.

Tobacco, alcohol, and human papillomavirus (HPV) are major risk factors for head and neck cancer (HNC), but it is unclear whether there are two distinct HNC risk groups, one associated with HPV and the other with tobacco/alcohol. Because HPV-positive HNC are clinically distinct from HPV-negative cases in treatment response and with more favorable prognoses, determining whether these differences result from infection alone or in association with other HNC risk factors is important for developing future therapeutic strategies. Incident cases of HNC (n = 201) and age-gender frequency-matched controls (n = 324) were recruited to assess anti-HPV VLP (virus like particles) antibodies 16, 18, 31, and 33.

Evidence for vertical transmission of HPV from mothers to infants.

Few large studies have evaluated concordance based on a broad spectrum of human papillomavirus (HPV) types in oral and genital specimens of mothers and their recently born infants. This information is important in determining whether HPV vaccines administered prior to pregnancy may be useful for preventing vertical transmission. HPV DNA was positive in 30% of mothers and 1.

Human papillomavirus, p16 and p53 expression associated with survival of head and neck cancer.

Background: P16 and p53 protein expression, and high-risk human papillomavirus (HPV-HR) types have been associated with survival in head and neck cancer (HNC). Evidence suggests that multiple molecular pathways need to be targeted to improve the poor prognosis of HNC. This study examined the individual and joint effects of tumor markers for differences in predicting HNC survival.

Interferon regulatory factor (IRF)-2 activates the HPV-16 E6-E7 promoter in keratinocytes.

Interferon regulatory factors (IRFs) are critical mediators of gene expression, cell growth and immune responses. We previously demonstrated that interferon (IFN) induction of early viral transcription and replication in several mucosal HPVs requires IRF-1 binding to a conserved interferon response element (IRE). Here we show that the IRF-2 protein serves as a baseline transactivator of the HPV-16 major early promoter, P97.

Risk factors and survival by HPV-16 E6 and E7 antibody status in human papillomavirus positive head and neck cancer.

High-risk human papillomavirus types (HPV-HR) are associated with head and neck cancer (HNC) risk and better survival. Most patients with HPV-HR DNA-positive tumors develop anti-HPV E6/E7 antibodies; however, it is unclear whether those who mount an immune response have similar risk factors or clinical outcomes as those who do not. HPV-16 DNA tumor-positive HNC cases were evaluated for HPV-16 E6 and E7 antibodies using a GST capture ELISA system.

Human papillomavirus (HPV) type 18 induces extended growth in primary human cervical, tonsillar, or foreskin keratinocytes more effectively than other high-risk mucosal HPVs.

Mucosal high-risk (HR) human papillomaviruses (HPVs) that cause cervical and other anogenital cancers also are found in approximately 25% of head and neck carcinomas (HNCs), especially those arising in the oropharynx and the tonsils. While many HR HPV types are common in anogenital cancer, over 90% of HPV-positive HNCs harbor HPV type 16 (HPV-16). Using a quantitative colony-forming assay, we compared the ability of full-length mucosal HPV genomes, i.

Cellular factor YY1 downregulates the human papillomavirus 16 E6/E7 promoter, P97, in vivo and in vitro from a negative element overlapping the transcription-initiation site.

Cellular factors that bind to cis sequences in the human papillomavirus 16 (HPV-16) upstream regulatory region (URR) positively and negatively regulate the viral E6 and E7 oncogene promoter, P97. DNase I footprinting has revealed the binding of cellular proteins to two previously undetected cis elements overlapping and 3' of the transcription-initiation site of the P97 promoter. Mutations within homologous motifs found in both of these cis elements abolished their negative function in vivo and the binding of the same cellular complex in vitro.

Interferon-beta treatment increases human papillomavirus early gene transcription and viral plasmid genome replication by activating interferon regulatory factor (IRF)-1.

Interferons (IFNs) have been used to treat mucosal lesions caused by human papillomavirus (HPV) infection, such as intraepithelial precursor lesions to cancer of the uterine cervix, genital warts or recurrent respiratory papillomatosis, to potentially reduce or eliminate replicating HPV plasmid genomes. Mucosal HPVs have evolved mechanisms that impede IFN-beta synthesis and downregulate genes induced by IFN. Here we show that these HPV types directly subvert a cellular transcriptional response to IFN-beta as a potential boost in infection.

Upstream regulatory region alterations found in human papillomavirus type 16 (HPV-16) isolates from cervical carcinomas increase transcription, ori function, and HPV immortalization capacity in culture.

Human papillomavirus (HPV) DNAs isolated from cervical and head and neck carcinomas frequently contain nucleotide sequence alterations in the viral upstream regulatory region (URR). Our study has addressed the role such sequence changes may play in the efficiency of establishing HPV persistence and altered keratinocyte growth. Genomic mapping of integrated HPV type 16 (HPV-16) genomes from 32 cervical cancers revealed that the viral E6 and E7 oncogenes, as well as the L1 region/URR, were intact in all of them.

Cellular factors are required to activate bovine papillomavirus-1 early gene transcription and to establish viral plasmid persistence but are not required for cellular transformation.

Transcription from the major upstream early gene promoter, P89, of bovine papillomavirus (BPV)-1 is detectable in transfected cells lacking viral gene products yet also responds to viral E2 proteins. In contrast to human papillomaviruses (HPVs), the BPV upstream regulatory region (URR) functions as a transcriptional enhancer in epithelial cells and fibroblasts of bovine, murine or human origin. Mutations of Sp1 and/or two novel transcriptional enhancer factor (TEF)-1 sites within the 5' URR of the intact BPV-1 genome dramatically reduced P89-initiated mRNA levels, leading to decreased BPV-1 plasmid amplification and inefficient formation of transformed cell foci.

Functional mapping of the human papillomavirus type 16 E1 cistron.

Replication of the double-stranded, circular human papillomavirus (HPV) genomes requires the viral DNA replicase E1. Here, we report an initial characterization of the E1 cistron of HPV type 16 (HPV-16), the most common oncogenic mucosal HPV type found in cervical and some head and neck cancers. The first step in HPV DNA replication is an initial burst of plasmid viral DNA amplification.

The E8--E2 gene product of human papillomavirus type 16 represses early transcription and replication but is dispensable for viral plasmid persistence in keratinocytes.

A conserved E8(wedge)E2 spliced mRNA is detected in keratinocytes transfected with human papillomavirus type 16 (HPV-16) plasmid DNA. Expression of HPV-16 E8--E2 (16-E8--E2) is independent of the major early promoter, P97, and is modulated by both specific splicing events and conserved cis elements in the upstream regulatory region in a manner that differs from transcriptional regulation of other early viral genes. Mutations that disrupt the predicted 16-E8--E2 message also increase initial HPV-16 plasmid amplification 8- to 15-fold and major early gene (P97) transcription 4- to 5-fold over those of the wild type (wt).

Does pretreatment seropositivity to human papillomavirus have prognostic significance for head and neck cancers?

Background: Human papillomavirus (HPV) is a risk factor for head and neck cancers (HNC), yet HPV-associated tumors have better prognosis than HPV-negative tumors.

Methods: We evaluated whether pretreatment presence of antibodies to HPV capsids [virus-like particles (VLP)] or to HPV-16 oncoproteins E6 and E7 was a predictor of HPV-positive HNC and clinical outcomes. Sera from 156 HNC patients were tested for antibodies to HPV-16-derived antigens using ELISA.

Association between p53 and human papillomavirus in head and neck cancer survival.

Background: High-risk human papillomavirus (HPV-HR) is a significant risk factor for head and neck cancer (HNC), abrogating normal p53 function. In addition, HPV and p53 have been associated with prognosis of these tumors but the findings have been inconsistent. We examined p53 expression and HPV-HR individually and jointly for differences in predicting HNC survival.

Prevalence of human papillomavirus in the oral cavity/oropharynx in a large population of children and adolescents.

Objective: Human papillomavirus (HPV) in the oral cavity or oropharynx is associated with an increased risk of laryngeal papillomatosis, head and neck cancer, and cervical and other genital cancers. We evaluated the prevalence of HPV DNA in the oral cavity/oropharynx in a cross section of children aged 2 weeks to 20 years.

Methods: A risk factor questionnaire and oral exfoliated cells were collected from children (N = 1235).

Fundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers.

Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells.

P16INK4a expression, human papillomavirus, and survival in head and neck cancer.

Development of head and neck cancer (HNC) is associated with human papillomavirus high-risk (HPV-HR) types. The HPV E7 oncoprotein inactivates the pRB protein increasing expression of p16(INK4a). P16 expression and HPV status have been associated with differences in clinical outcomes for HNC.