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. Furthermore, relative miRNAs abundance levels of primary and immortalized human keratinocyte clones were evaluated. The global quantitation of miRNA gene abundance was performed using a TaqMan Low Density Array system. The confirmation of differentially expressed miRNAs was performed on a set of formalin-fixed paraffin-embedded tumor samples enriched for the tumor cell fraction by macrodissection. RESULTS: We defined 46 upregulated and 31 downregulated miRNAs characteristic for the HPV-positive tonsillar tumors and 42 upregulated miRNAs and 42 downregulated miRNAs characteristic for HPV-independent tumors. In comparison with the expression profiles in cervical tumors, we defined miR-141-3p, miR-15b-5p, miR-200a-3p, miR-302c-3p, and miR-9-5p as specific for HPV induced malignancies. MiR-335-5p, miR-579-3p, and miR-126-5p were shared by the expression profiles of HPV-positive tonsillar tumors and of the HPV immortalized keratinocyte clones, whereas miR-328-3p, miR-34c-3p, and miR-885-5p were shared by the miRNA profiles of HPV-negative tonsillar tumors and the HPV-negative keratinocytes. CONCLUSIONS: We identified the miRNAs characteristic for HPV-induced tumors and tonsillar tumors of different etiology, and the results were compared with those of the model system. Our report presents the basis for further investigations leading to the identification of clinically relevant diagnostic and/or therapeutic biomarkers for tumors of viral and non-viral etiology.

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). In this study, we have characterised the physical status of HPV in a set of tonsillar tumour samples using different methods--the mapping of E2 integration breakpoint at the mRNA level, the 3' RACE based Amplification of Papillomavirus Oncogene Transcripts (APOT) assay and Southern blot. Furthermore, the impact of HPV integration on patients' prognosis has been evaluated in a larger set of 186 patients with head and neck cancer. Based on the analysis of E2 mRNA, HPV was integrated in the host genome in 43% of the HPV-positive samples. Extrachromosomal or mixed form was present in 57%. In fresh frozen samples, the APOT and E2 mapping results were in agreement. The results were confirmed using Southern blotting. Furthermore, the type and exact site of integration were determined. The survival analysis of 186 patients revealed HPV positivity, tumour size and lymph node positivity as factors that influence disease specific survival. However, no statistically significant difference was found in disease specific survival between patients with HPV-positive integrated vs. extrachromosomal/mixed forms of the virus.

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. However, treated cultures eventually gave rise to outgrowth of clones harboring integrated HPV-16 genomes expressing viral E6 and E7 oncogenes from chimeric virus-cell mRNAs similar to those in cervical and head and neck cancers. Surprisingly, every HPV-16 integrant that arose after IFN exposure stemmed from an independent integration event into a different cellular gene locus, even within parallel cultures started from small cell inocula and cultured separately for ≥ 25 doublings to permit the rise and expansion of spontaneous integrants. While IFN treatment conferred a growth advantage upon preexisting integrants added to mixed control cultures, our results indicate that IFN exposure directly or indirectly induces HPV-16 integration, rather than only selects preexisting, spontaneous integrants that appear to be much less frequent. We estimate that IFN exposure increased integration rates by ≥ 100-fold. IFN-induced HPV-16 integration involved a wide range of chromosomal loci with less apparent selection for recurrent insertions near genes involved in cancer-related pathways. We conclude that IFNs and other potential treatments targeting high-risk HPV persistence that disrupt viral genome replication may promote increased high-risk HPV integration as a step in cancer progression. Therapies against high-risk HPV persistence thus need to be evaluated for their integration-inducing potential.

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. In conjunction with additional methods, these protocols enable examination of genomic stability, viral and cellular gene expression, viral integration, and differentiation patterns influenced by HPV persistence in clonal human keratinocytes that effectively mimic early events in HPV infection.

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

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. Here we show that E2-TR trans-activates E2-dependent promoters 5 to 10-fold in cooperation with cellular factors and in a dose-dependent fashion in epithelial cells and fibroblasts of animal or human origin while E2-TA activated >100-fold and the E8^E2 had no effect. However, in contrast to E2-TA, E2-TR activated transcription from a promoter-proximal position. E2-TR also partially inhibited the BPV-1 P89 or heterologous promoters whereas E8^E2 led to complete repression. Thus, the BPV-1 E2-TR modulates viral gene expression in a manner distinct from other E2 proteins.

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 = 1.4); and HPV-seropositive/heavy alcohol users (adjusted OR = 9.8) had greater risk than HPV-seronegative/heavy alcohol users (adjusted OR = 3.1). In contrast, the risk of oropharyngeal cancer was greater in the HPV-seronegative/heavy tobacco (adjusted OR = 11.0) than in HPV-seropositive/heavy tobacco (adjusted OR = 4.7) users and greater in HPV-seronegative/heavy alcohol users (adjusted OR = 24.3) compared to HPV-seropositive/heavy alcohol users (adjusted OR = 8.5). Disease-specific and recurrence-free adjusted survival were significantly worse in oropharyngeal HPV-seronegative cases with no survival differences by HPV status seen in oral cavity cases. The association between tobacco/alcohol, HPV, and tumor site is complex. There appear to be distinct tumor site differences in the combined exposure risks, suggesting that different molecular pathways are involved.

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). RESULTS: HPV-16 E6 and/or E7 seropositive HNC cases were associated with higher risk in younger patients (≤ 55 years), more sexual partners (≥ 10), oropharyngeal cancer, worse stage at diagnosis, poorer grade, and nodal involvement. Between BL and FU (median = 8.3 months), there were decreased antibody levels for seropositive E6 (73% vs. 27%, p = 0.02) and seropositive E7 patients (65% vs. 35%, p = 0.09) with 5% of BL E6 and 35% of BL E7 seropositive patients converting to negative status at FU. Overall mortality (OM) was significantly worse among BL E6 seronegative patients than among BL seropositive patients (40.2% vs.13.6%, p = 0.01). There were no disease specific (DS) deaths among BL E6 seropositive vs. 24% in BL E6 seronegative patients (p = 0.01). BL E7 seronegative patients also had higher mortality than BL seropositive patients (OM: 38.2% vs. 20.0%, p = 0.04; DS: 22.5% vs. 5.6%, p = 0.07). CONCLUSION: These findings are the first to follow post-treatment OD levels of HPV-16 E6 and E7 in HNC and suggest that these HPV antibodies may be potential prognostic markers of survival in HNC patients.

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. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.

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. Multivariate logistic regression and stratified analyses were used to calculate adjusted odds ratios (OR). HPV-seronegative and seropositive/heavy tobacco users had similar increased adjusted risks of HNC (HPV-seronegative OR = 2.6, 1.4-5.0; HPV-seropositive OR = 2.3, 1.1-4.8), as did HPV-seronegative (OR = 4.3, 2.1-9.1) versus HPV-seropositive/heavy alcohol users (OR = 3.9, 1.6-9.4). Similar HPV/tobacco/alcohol risk profiles also were seen in oropharyngeal and oral cavity tumor sites. Our finding that tobacco/alcohol use increased the risk of HNC in both HPV-seropositive and HPV-seronegative individuals is consistent with the observation that HPV infection is not a sufficient cause of HNC but requires the accumulation of additional cellular changes.

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 1.5% of newborns. Maternal/newborn concordance (HPV+/+ or HPV-/-) was 71%. Among HPV DNA+ mothers, only 3% of their infants were DNA+ and only 1 pair had the same HPV type. Among HPV- women, 0.8% of infants were HPV+. HPV DNA detected in hospitalized newborns reflects current infection transmitted to infants during pregnancy or delivery. None of the mother/baby HPV DNA+ concordance pairs detected viral types found in HPV vaccines suggesting that vaccination prior to pregnancy is unlikely to be efficacious in preventing vertical transmission.

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. Cotransfections in IRF knockout cells confirmed that basal HPV-16 promoter activity was supported by both IRF-1 and IRF-2 complexes interacting with the promoter-proximal IRE in a dose-dependent manner. Furthermore, HPV-16 E7 expression downregulates the IRF-2 promoter, thus linking IRF-2 levels to viral transforming gene expression through a negative feedback mechanism. Taken together, these observations reveal a complex viral strategy utilizing multiple signal transduction pathways during the establishment and maintenance of HPV persistence.

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. P16 and p53 expression were detected from formalin-fixed, paraffin-embedded tissues by immunohistochemical staining. HPV DNA was detected by PCR and DNA sequencing in 237 histologically confirmed HNC patients. RESULTS: Overexpression of p16 (p16+) and p53 (p53+) occurred in 38% and 48% of HNC tumors, respectively. HPV-HR was detected in 28% of tumors. Worse prognosis was found in tumors that were p53+ (disease-specific mortality: adjusted hazard ratios, HR = 1.9, 95% CI: 1.04-3.4) or HPV- (overall survival: adj. HR = 2.1, 1.1-4.3) but no association in survival was found by p16 status. Compared to the molecular marker group with the best prognosis (p16+/p53-/HPV-HR: referent), the p16-/p53+/HPV- group had the lowest overall survival (84% vs. 60%, p < 0.01; HR = 4.1, 1.7-9.9) and disease-specific survival (86% vs. 66%, p < 0.01; HR = 4.0, 1.5-10.7). Compared to the referent, the HRs of the other six joint biomarker groups ranged from 1.6-3.4 for overall mortality and 0.9-3.9 for disease-specific mortality. CONCLUSION: The p16/p53/HPV joint groups showed greater distinction in clinical outcomes compared to results based on the individual biomarkers alone. This finding suggests that assessing multiple molecular markers in HNC patients will better predict the diverse outcomes and potentially the type of treatment targeted to those markers.

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. Among 57 HPV-16 DNA tumor-positive HNC cases, 67% were detected with HPV-16 E6 and/or E7 antibodies. Male gender (76% vs. 42%, p = 0.02), younger age (63% vs. 16%, p = 0.001) but not tobacco or alcohol were associated with E6 and/or E7 seropositivity. Seropositivity was associated more often with late stage (76%), poor grade (65%), positive nodes (82%). and in the oropharynx (82%), Median disease-specific and recurrence-free survival were longer in E6 and/or E7 seropositive compared to E6/E7-negative cases (2.2 years vs. 1.4 years, both outcomes), although results were not statistically significant. When examined jointly with p16 expression, E6 and/or E7-positive/p16-positive cases had better disease-specific (2.1 years vs. 1.1 years, p = 0.06) and recurrence-free (2.3 years vs. 1.1 years, p = 0.03) survival compared to E6-/E7-/p16- cases. These findings suggest there are 2 distinct HNC patient groups with HPV DNA-positive tumors, distinguishable by E6 and/or E7 antibody status. Differences in antibody status are associated with distinct risk factors and clinical outcomes. This information can be available as a simple blood test at initial presentation, before the removal of tissue through biopsy or surgery.

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.e., the low-risk HPV-11 and HR HPV-16, -18, and -31, to persist in and alter the growth of primary human keratinocytes from the foreskin, cervix, and tonsils. The HR HPV types led to the formation of growing keratinocyte colonies in culture independent of the site of epithelial origin. However, HPV-18 induced colony growth in all keratinocytes >4-fold more effectively than HPV-16 or HPV-31 and >20-fold more efficiently than HPV-11 or controls. HPV-11-transfected or control colonies failed to expand beyond 32 to 36 population doublings postexplantation. In contrast, individual HR HPV-transfected clones exhibited no apparent slowdown of growth or "crisis," and many maintained HPV plasmid persistence beyond 60 population doublings. Keratinocyte clones harboring extrachromosomal HR HPV genomes had shorter population doubling times and formed dysplastic stratified epithelia in organotypic raft cultures, mirroring the pathological features of higher-grade intraepithelial lesions, yet did not exhibit chromosomal instability. We conclude that, in culture, the HR HPV type, rather than the site of epithelial origin of the cells, determines the efficacy of inducing continued growth of individual keratinocytes, with HPV-18 being the most aggressive mucosal HR HPV type tested.

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. This factor was identified as YY1 by complex mobility and binding specificity in comparison with vaccinia virus-expressed, purified recombinant YY1 protein and by antigenic reactivity with YY1 antisera. Cis mutations in the 'initiator' YY1 site activated the P97 promoter in vivo and in vitro. P97 was also activated threefold in vitro by depletion of endogenous YY1 with wild-type, but not mutant, YY1 oligonucleotides from the IgH kappa E3' enhancer. Furthermore, increasing concentrations of exogenous, purified recombinant YY1 repressed wild-type P97 transcript levels by up to threefold, but did not influence the P97 promoter mutated in the 'initiator' YY1 site. Thus, the promoter-proximal YY1 site was not necessary for correct transcription initiation at the P97 promoter, but was found to be required for downregulation of P97 transcription in vivo and in vitro. In contrast to other viral and cellular promoters, where YY1 is thought to function as a positive transcription-'initiator' factor, HPV-16 P97 transcription is downregulated by YY1 from a critical motif overlapping the transcription start site.

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. Treatment with low levels of human IFN-beta induced initial amplification of HPV-16 and HPV-11 plasmid genomes and increased HPV-16 or HPV-31 DNA copy numbers up to 6-fold in HPV-immortalized keratinocytes. IFN treatment also increased early gene transcription from the major early gene promoters in HPV-16, HPV-31 and HPV-11. Furthermore, mutagenesis of the viral genomes and ectopic interferon regulatory factor (IRF) expression in transfection experiments using IRF-1(-/-), IRF-2(-/-) and dual knockout cell lines determined that these responses are due to the activation of IRF-1 interaction with a conserved interferon response element demonstrated in several mucosal HPV early gene promoters. Our results provide a molecular explanation for the varying clinical outcomes of IFN therapy of papillomatoses and define an assay for the modulation of the HPV gene program by IFNs as well as other cytokines and signaling molecules in infection and therapy.

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. The URR sequences from integrated and unintegrated viral DNA were found to harbor distinct sets of nucleotide substitutions. A subset of the altered URRs increased the potential of HPV-16 to establish persistent, cell growth-altering viral-genome replication in the cell. This aggressive phenotype in culture was not solely due to increased viral early gene transcription, but also to augmented initial amplification of the viral genome. As revealed in a novel ori-dependent HPV-16 plasmid amplification assay, the altered motifs that led to increased viral transcription from the intact genome also greatly augmented HPV-16 ori function. The nucleotide sequence changes correlate with those previously described in the distinct geographical North American type 1 and Asian-American variants that are associated with more aggressive disease in epidemiologic studies and encompass, but are not limited to, alterations in previously characterized sites for the negative regulatory protein YY1. Our results thus provide evidence that nucleotide alterations in HPV regulatory sequences could serve as potential prognostic markers of HPV-associated carcinogenesis.

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. However, cell lines transformed with wt or mutant BPV-1 genomes contained similar levels of unintegrated BPV-1 DNA, P89 mRNA and E2-dependent transactivation. We conclude that cellular factors necessary for activating viral early gene transcription, establishment of viral plasmid replication and cell immortalization are not required during the maintenance phase of BPV-1 infection.

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. HPV-16 in tumors was evaluated by PCR and DNA sequencing. RESULTS: HPV-16 antibodies were found in 33% with HPV-16 VLP, 21% with HPV-16 E6, and 21% with E7. HPV-16 was detected in 26% of tumors. There was a strong correlation between detection of HPV-16 tumor DNA and antibodies to HPV-16 E6 or E7 (kappa = 0.7) but not to HPV-16 VLP (kappa = 0.4). Multivariate analyses showed significantly better disease-specific survival in seropositive HPV-16 VLP [hazard ratio (HR), 0.4; 95% confidence interval (95% CI), 0.1-0.9], HPV-16 E6 (HR, 0.1; 95% CI, 0.02-0.5), and HPV-16 E7 (HR, 0.3; 95% CI, 0.1-0.9) cases. Less disease recurrence occurred among those with antibodies to both E6 and E7 compared with those negative to both (P = 0.003). There was better disease-specific survival in patients who were E6 positive at baseline and remained positive at follow-up compared with individuals who were E6 negative at both time points (P = 0.03; kappa = 0.9). CONCLUSIONS: The presence of antibodies to HPV-16 E6 and E7 is associated with HPV in tumor cells and with better clinical outcomes. These findings suggest that the presence of E6/E7 antibodies before treatment is predictive of better clinical outcomes and that they may serve as biomarkers for selecting targeted therapeutic modalities developed for HPV-associated tumors.

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). Expressing the 16-E8--E2 gene product from the cytomegalovirus (CMV) promoter represses HPV-16 early gene transcription from P97 in a dose-dependent manner, as detected by RNase protection assays. When expressed from the CMV promoter, 16-E8--E2 also inhibits the amplification of an HPV-16 plasmid and a heterologous simian virus 40 (SV40) ori plasmid that contains E2 binding sites in cis. In contrast, cotransfections with HPV-16 wt genomes that express physiologic levels of 16-E8--E2 are sufficient to repress HPV-16 plasmid amplification but are limiting and insufficient for the repression of SV40 amplification. 16-E8--E2-dependent repression of HPV-16 E1 expression is sufficient to account for this observed inhibition of initial HPV-16 plasmid amplification. Unlike with other papillomaviruses, primary human keratinocytes immortalized by the HPV-16 E8 mutant genome contain more than eightfold-higher levels of unintegrated plasmid than the wt, demonstrating that 16-E8(wedge)E2 limits the viral copy number but is not required for plasmid persistence and maintenance.