Silver nanoparticles exhibit size-dependent differential toxicity and induce expression of syncytin-1 in FA-AML1 and MOLT-4 leukaemia cell lines.

Human endogenous retrovirus (HERV) sequences make up ~8% of the human genome and increased expression of some HERV proteins has been observed in various pathologies including leukaemia and multiple sclerosis. However, little is known about the function of these HERV proteins or environmental factors which regulate their expression. Silver nanoparticles (AgNPs) are used very extensively as antimicrobials and antivirals in numerous consumer products although their effect on the expression of HERV gene products is unknown. Cell proliferation and cell toxicity assays were carried out on human acute T lymphoblastic leukaemia (MOLT-4) and Fanconi anaemia associated acute myeloid leukaemia (FA-AML1) cells treated with two different sizes of AgNPs (7nm and 50nm diameter). Reverse-transcriptase polymerase chain reaction and western blotting were then used to the assess expression of HERV-W syncytin-1 mRNA and protein in these cells. FA-AML1 cells were more sensitive overall than MOLT-4 to treatment with the smaller 7nm sized AgNp's being the most toxic in these cells. MOLT-4 cell were more resistant and showed no evidence of differential toxicity to the different sized particles. Syncytin-1 mRNA and protein were induced by both 7 and 50nm AgNPs in both cell types yet with different kinetics. In summary, the observation that AgNPs induce expression of syncytin-1 in FA-AML1 and MOLT-4 cells at doses as little as 5 µg/ml is grounds for concern since this protein is up-regulated in both malignant and neurodegenerative diseases. Considering the widespread use of AgNPs in the environment it is clear that their ability to induce syncytin-1 should be investigated further in other cell types.

Increased prevalence of JC polyomavirus in cervical carcinomas from women infected with HIV.

Although subclinical persistent infections with the human polyomaviruses BKV and JCV are ubiquitous worldwide, these are known to vary in relation to diseases present and geographical location. DNAs from 220 cervical smears and 109 invasive cervical carcinomas obtained from HIV positive and HIV negative Kenyan women of known HPV status were analyzed by nested endpoint PCR for BKV and JCV. BKV-JCV DNA was detected in 5/105 (4.7%) of cervical smears and in 6/37 (16%) of cervical carcinomas from women infected with HIV whereas 9/115 (7.8%) of the cervical smears and 4/72 (5.5%) of the carcinomas were positive in HIV negative women. Nested PCR showed that all 24 samples were positive for JCV and not BKV. JCV was not more prevalent in either HPV positive (P = 0.438) or HPV negative women (P = 0.392). However, 37% of carcinomas and smears which were positive for JCV were also positive for a "high-risk" oncogenic HPV. Comparison of the incidence of JCV in cervical smears and cervical carcinomas showed a ∼3-fold increase in samples from HIV positive women with cervical carcinoma (P = 0.025) whereas no significant difference was found between cervical smears and cervical carcinomas from HIV negative women (P = 0.553). These results suggest that JCV may combine with high-risk HPV infection in women infected with HIV to influence the rate of progression to invasive cervical carcinoma.

Donor KIR2DL1 Allelic Polymorphism Influences Posthematopoietic Progenitor Cell Transplantation Outcomes in the T Cell Depleted and Reduced Intensity Conditioning Setting.

The majority of established KIR clinical assessment algorithms used for donor selection for hematopoietic progenitor cell transplantation (HPCT) evaluate gene content (presence/absence) of the KIR gene complex. In comparison, relatively little is known about the impact of KIR allelic polymorphism. By analyzing donors of T cell depleted (TcD) reduced intensity conditioning (RIC) HPCT, this study investigated the influence on post-transplant outcome of 2 polymorphic residues of the inhibitory KIR2DL1. The aim of this study was to expand upon existing research into the influence of KIR2DL1 allelic polymorphism upon post-transplant outcome. The effects of allele groups upon transplant outcomes were investigated within a patient cohort using a defined treatment protocol of RIC with TcD. Using phylogenetic data, KIR2DL1 allelic polymorphism was categorized into groups on the basis of variation within codons 114 and 245 (positive or negative for the following groups: KIR2DL1*002/001g, KIR2DL1*003, KIR2DL1*004g) and the identification of null alleles. The influence of these KIR2DL1 allele groups in hematopoietic progenitor cell transplantation (HPCT) donors was assessed in the post-transplant data of 86 acute myelogenous leukemia patients receiving RIC TcD HPCT at a single center. KIR2DL1 allele groups in the donor significantly impacted upon 5-year post-transplant outcomes in RIC TcD HPCT. Donor KIR2DL1*003 presented the greatest influence upon post-transplant outcomes, with KIR2DL1*003 positive donors severely reducing 5-year post-transplant overall survival (OS) compared to those receiving a transplant from a KIR2DL1*003 negative donor (KIR2DL1*003 pos versus neg: 27.0% versus 60.0%, P = .008, pc = 0.024) and disease-free survival (DFS) (KIR2DL1*003 pos versus neg: 23.5% versus 60.0%, P = .004, pc = 0.012), and increasing 5-year relapse incidence (KIR2DL1*003 pos versus neg: 63.9% versus 27.2%, P = .009, pc = 0.027). KIR2DL1*003 homozygous and KIR2DL1*003 heterozygous grafts did not present significantly different post-transplant outcomes. Donors possessing the KIR2DL1*002/001 allele group were found to significantly improve post-transplant outcomes, with donors positive for the KIR2DL1*004 allele group presenting a trend towards improvement. KIR2DL1*002/001 allele group (KIR2DL1*002/001g) positive donors improved 5-year OS (KIR2DL1*002/001g pos versus neg: 56.4% versus 27.2%, P = .009, pc = 0.024) and DFS (KIR2DL1*002/001g pos versus neg: 53.8% versus 25.5%, P = .018, pc = 0.036). KIR2DL1*004 allele group (KIR2DL1*004g) positive donors trended towards improving 5-year OS (KIR2DL1*004g pos versus neg: 53.3% versus 35.5%, P = .097, pc = 0.097) and DFS (KIR2DL1*004g pos versus neg: 50.0% versus 33.9%, P = .121, pc = 0.121), and reducing relapse incidence (KIR2DL1*004g pos versus neg: 33.1% versus 54.0%, P = .079, pc = 0.152). The presented findings suggest donor selection algorithms for TcD RIC HPCT should consider avoiding KIR2DL1*003 positive donors, where possible, and contributes to the mounting evidence that KIR assessment in donor selection algorithms should reflect the conditioning regime protocol used.

Plexin D1: new potential biomarker for cervical cancer.

To explore possible role of Plexin D1 in cancer angiogenesis with special focus on cervical cancer. Twelve various normal tissues, 12 various tumor samples, and 59 different stages of cervical cancer samples on tissue microarrays were examined for the expression of Plexin D1. The findings of our study clearly indicate that Plexin D1 is strongly associated with cellular differentiation in the tissues investigated, and that expression is strongly dependent on the tumor histotype. In some tumor subtypes, the protein was detected at several-fold higher levels than was found in the corresponding normal tissues, while in others, expression was similar to normal tissues. Most significantly, strong expression was detected in the endothelial cells of the cervical cancer samples, yet no expression was seen in endothelial cells of normal cervical tissues, which suggests a potential role of Plexin D1 in cervical cancer-associated angiogenesis.Regarding the implications of Plexin D1 and its associations with cancer angiogenesis, it might be a potential cervical cancer biomarker if further studies confirm the present preliminary findings.

Analysis of human total antibody repertoires in TIF1γ autoantibody positive dermatomyositis.

We investigate the accumulated microbial and autoantigen antibody repertoire in adult-onset dermatomyositis patients sero-positive for TIF1γ (TRIM33) autoantibodies. We use an untargeted high-throughput approach which combines immunoglobulin disease-specific epitope-enrichment and identification of microbial and human antigens. We observe antibodies recognizing a wider repertoire of microbial antigens in dermatomyositis. Antibodies recognizing viruses and Poxviridae family species are significantly enriched. The identified autoantibodies recognise a large portion of the human proteome, including interferon regulated proteins; these proteins cluster in specific biological processes. In addition to TRIM33, we identify autoantibodies against eleven further TRIM proteins, including TRIM21. Some of these TRIM proteins share epitope homology with specific viral species including poxviruses. Our data suggest antibody accumulation in dermatomyositis against an expanded diversity of microbial and human proteins and evidence of non-random targeting of specific signalling pathways. Our findings indicate that molecular mimicry and epitope spreading events may play a role in dermatomyositis pathogenesis.

Combining metabolic fingerprinting and footprinting to understand the phenotypic response of HPV16 E6 expressing cervical carcinoma cells exposed to the HIV anti-viral drug lopinavir.

Recently, it has been reported that the anti-viral drug, lopinavir, which is currently used as a human immunodeficiency virus (HIV) protease inhibitor, could also inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells. In this study, C33A parent control cells and HPV16 E6-transfected cells were exposed to lopinavir at concentrations ranging from 0 to 30 microM. The phenotypic response was assessed by Fourier transform infrared (FT-IR) spectroscopy directly on cells (the metabolic fingerprint) and on the cell growth medium (the metabolic footprint). Multivariate analysis of the data using both principal components analysis (PCA) and canonical variates analysis (PC-CVA) showed trends in scores plots that were related to the concentration of the drug. Inspection of the PC-CVA loadings vector revealed that the effect was not due to the drug alone and that several IR spectral regions including proteins, nucleotides and carbohydrates contributed to the separation in PC-CVA space. Finally, partial least squares regression (PLSR) could be used to predict the concentration of the drug accurately from the metabolic fingerprints and footprints, indicating a dose related phenotypic response. This study shows that the combination of metabolic fingerprinting and footprinting with appropriate chemometric analysis is a valuable approach for studying cellular responses to anti-viral drugs.

Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells.

It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 µM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.

Potential Effects of Human Papillomavirus Type Substitution, Superinfection Exclusion and Latency on the Efficacy of the Current L1 Prophylactic Vaccines.

There are >200 different types of human papilloma virus (HPV) of which >51 infect genital epithelium, with the ~14 of these classed as high-risk being more commonly associated with cervical cancer. During development of the disease, high-risk types have an increased tendency to develop a truncated non-replicative life cycle, whereas low-risk, non-cancer-associated HPV types are either asymptomatic or cause benign lesions completing their full replicative life cycle. HPVs can also be present as non-replicative so-called "latent" infections and they can also show superinfection exclusion, where cells with pre-existing infections with one type cannot be infected with a different HPV type. Thus, the HPV repertoire and replication status present in an individual can form a complex dynamic meta-community which changes with respect to both time and exposure to different HPV types. In light of these considerations, it is not clear how current prophylactic HPV vaccines will affect this system and the potential for iatrogenic outcomes is discussed in light of recent outcome data.

Potential risk factors associated with the use of cidofovir to treat benign human papillomavirus-related disease.

BACKGROUND: Cidofovir is currently being used off-licence to treat different viral infections, such as benign low-risk human papillomavirus (HPV)-related recurrent respiratory papillomatosis (RRP). There are concerns over the safety of this practice as rat studies demonstrated a high malignant transformation rate. As yet, there are no clinical reports of cidofovir-induced malignant changes in humans. METHODS: Telomerase immortalised human keratinocytes (hTert) stably expressing E6 proteins from either low-risk HPV6b or high-risk HPV16 and vector control cells were treated with either low-dose (5 microg/ml) or higher dose (30 microg/ml) cidofovir for 2 days and the effects evaluated by clonogenic survival assays. Based on these results, gene expression microarray analysis was performed on cidofovir-treated low-risk E6 and vector cells before, during and after drug treatment, and the results verified by real-time PCR. RESULTS: Both low-risk and high-risk E6-expressing cells show significantly improved long-term survival compared with vector control cells when exposed to 5 microg/ml cidofovir for 2 days, (hTert T6E6 P=0.0007, hTert T16E6 P=0.00023 and hTert vector control P=0.62). Microarray and real-time PCR analyses of low-dose cidofovir-treated low-risk E6-expressing cells revealed changes in gene expression that are known to be associated with malignant progression, which were not observed in drug-treated vector control cells. CONCLUSIONS: This is the first report that cidofovir can both increase cell survival and induce alterations in gene expression that are known to be associated with malignant transformation in cells transduced only with the E6 gene from low-risk HPV. It is our belief that these data provide cause for concern over the off-license use of this drug to treat RRP.

Analysis of head and neck carcinoma progression reveals novel and relevant stage-specific changes associated with immortalisation and malignancy.

We report changes in the genomic landscape in the development of head and neck squamous cell carcinomas HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Likely pathological mutations predominantly involved a relatively small set of genes reported previously (TP53, KMT2D, CDKN2A, PIK3CA, NOTCH1 and FAT1) but also other predicted cancer drivers (MGA, PABPC3, NR4A2, NCOR1 and MACF1). Notably, all these mutations arise early and are present in PPOLs. The most frequent genetic changes, which follow acquisition of immortality and loss of senescence, are of consistent somatic copy number alterations (SCNAs) involving chromosomal regions enriched for genes in known and previously unreported cancer-related pathways. We mapped the evolution of SCNAs in HNSCC progression. One of the earliest SCNAs involved deletions of CSMD1 (8p23.2). CSMD1 deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines. Modulation of CSMD1 in cell lines revealed significant suppression of proliferation and invasion by forced expression, and significant stimulation of invasion by knockdown of expression. Known cancer drivers NOTCH1, PPP6C, RAC1, EIF4G1, PIK3CA showed significant increase in frequency of SCNA in transition from PPOLs to HNSCC that correlated with their expression. In the later stages of progression, HNSCC with and without nodal metastases showed some clear differences including high copy number gains of CCND1, hsa-miR-548k and TP63 in the metastases group.

Tumor expression of major vault protein is an adverse prognostic factor for radiotherapy outcome in oropharyngeal carcinoma.

PURPOSE: Vaults are multi-subunit structures that may be involved in nucleo-cytoplasmic transport, with the major vault protein (MVP or lung resistance-related protein [LRP]) being the main component. The MVP gene is located on chromosome 16 close to the multidrug resistance-associated protein and protein kinase c-beta genes. The role of MVP in cancer drug resistance has been demonstrated in various cell lines as well as in ovarian carcinomas and acute myeloid leukemia, but nothing is known about its possible role in radiation resistance. Our aim was to examine this in head-and-neck squamous cell carcinoma (HNSCC). METHODS AND MATERIALS: Archived biopsy material was obtained for 78 patients with squamous cell carcinoma of the oropharynx who received primary radiotherapy with curative intent. Immunohistochemistry was used to detect MVP expression. Locoregional failure and cancer-specific survival were estimated using cumulative incidence and Cox multivariate analyses. RESULTS: In a univariate and multivariate analysis, MVP expression was strongly associated with both locoregional failure and cancer-specific survival. After adjustment for disease site, stage, grade, anemia, smoking, alcohol, gender, and age, the estimated hazard ratio for high MVP (2/3) compared with low (0/1) was 4.98 (95% confidence interval, 2.17-11.42; p = 0.0002) for locoregional failure and 4.28 (95% confidence interval, 1.85-9.95; p = 0.001) for cancer-specific mortality. CONCLUSION: These data are the first to show that MVP may be a useful prognostic marker associated with radiotherapy resistance in a subgroup of patients with HNSCC.

Specific HIV protease inhibitors inhibit the ability of HPV16 E6 to degrade p53 and selectively kill E6-dependent cervical carcinoma cells in vitro.

Although HIV protease inhibitor (PI) drugs predominantly target HIV proteases 1 and 2, it is also known that part of their efficacy is due to selective inhibition of the proteasome. The pathogenicity of high-risk human papilloma virus (HPV) is dependent on expression of viral E6 proteins which inappropriately activate the 26S proteasome to degrade p53 and other cellular proteins that are detrimental to viral replication. Comparison of the ability of the PIs indinavir, ritonavir, amprenavir, lopinavir, atazanavir, nelfinavir and saquinavir to inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells showed that 15 microM lopinavir, 1 mM indinavir or 125 microM ritonavir treatment for 24 h produced a stable increase in the level of nuclear p53 in these cells with minimal cell death. After 4 h exposure of HPV16+ve SiHa cells to 15 microM lopinavir, a transient increase in wild-type p53 expression was observed associated with a 7% reduction in the chymotryptic activity of the 205 proteasome and apoptosis after 24h. Comparison of growth rates of PI treated SiHa, CaSki, C33A, C33A-E6 and non-transformed NIH/3T3 cells showed that SiHa were the most sensitive, whereas NIH/3T3 were least affected. In conclusion, these data show that specific HIV PIs such as lopinavir and possibly indinavir, can induce selective toxicity of HPV-transformed cervical carcinoma cells expressing wild-type p53 and may form the basis of a topically applied alternative to surgery for the treatment of HPV-related premalignant lesions of the cervix.

Hypoxia increases heparanase-dependent tumor cell invasion, which can be inhibited by antiheparanase antibodies.

The beta-endoglucuronidase heparanase plays an important role in tumor invasion, a process that is significantly enhanced by hypoxia. We have used a strategy of stable transfection with antisense to derive ovarian carcinoma cell lines that express different levels of heparanase and used these to demonstrate that invasion correlates with heparanase activity. Secreted heparanase activity was increased by reduction, hypoxia, and growth of cells under reduced oxygen (1%) augmented heparanase activity and invasion, both of which are inhibited by treatment with antiheparanase antibodies. This is the first demonstration that heparanase activity may be regulated by microenvironmental redox conditions, which influence invasion, and that invasion can be blocked with specific heparanase-neutralizing antibodies.

Analysis of the Prevalence of HTLV-1 Proviral DNA in Cervical Smears and Carcinomas from HIV Positive and Negative Kenyan Women.

The oncogenic retrovirus human T-cell lymphotropic virus type 1 (HTLV-1) is endemic in some countries although its prevalence and relationship with other sexually transmitted infections in Sub-Saharan Africa is largely unknown. A novel endpoint PCR method was used to analyse the prevalence of HTLV-1 proviral DNA in genomic DNA extracted from liquid based cytology (LBC) cervical smears and invasive cervical carcinomas (ICCs) obtained from human immunodeficiency virus-positive (HIV+ve) and HIV-negative (HIV-ve) Kenyan women. Patient sociodemographic details were recorded by structured questionnaire and these data analysed with respect to HIV status, human papillomavirus (HPV) type (Papilocheck(®)) and cytology. This showed 22/113 (19.5%) of LBC's from HIV+ve patients were positive for HTLV-1 compared to 4/111 (3.6%) of those from HIV-ve women (p = 0.0002; odds ratio (OR) = 6.42 (2.07-26.56)). Only 1/37 (2.7%) of HIV+ve and none of the 44 HIV-ve ICC samples were positive for HTLV-1. There was also a significant correlation between HTLV-1 infection, numbers of sexual partners (p < 0.05) and smoking (p < 0.01). Using this unique method, these data suggest an unexpectedly high prevalence of HTLV-1 DNA in HIV+ve women in this geographical location. However, the low level of HTLV-1 detected in HIV+ve ICC samples was unexpected and the reasons for this are unclear.

A Single-Arm, Proof-Of-Concept Trial of Lopimune (Lopinavir/Ritonavir) as a Treatment for HPV-Related Pre-Invasive Cervical Disease.

BACKGROUND: Cervical cancer is the most common female malignancy in the developing nations and the third most common cancer in women globally. An effective, inexpensive and self-applied topical treatment would be an ideal solution for treatment of screen-detected, pre-invasive cervical disease in low resource settings. METHODS: Between 01/03/2013 and 01/08/2013, women attending Kenyatta National Hospital's Family Planning and Gynaecology Outpatients clinics were tested for HIV, HPV (Cervista®) and liquid based cervical cytology (LBC-ThinPrep®). HIV negative women diagnosed as high-risk HPV positive with high grade squamous intraepithelial lesions (HSIL) were examined by colposcopy and given a 2 week course of 1 capsule of Lopimune (CIPLA) twice daily, to be self-applied as a vaginal pessary. Colposcopy, HPV testing and LBC were repeated at 4 and 12 weeks post-start of treatment with a final punch biopsy at 3 months for histology. Primary outcome measures were acceptability of treatment with efficacy as a secondary consideration. RESULTS: A total of 23 women with HSIL were treated with Lopimune during which time no adverse reactions were reported. A maximum concentration of 10 ng/ml of lopinavir was detected in patient plasma 1 week after starting treatment. HPV was no longer detected in 12/23 (52.2%, 95%CI: 30.6-73.2%). Post-treatment cytology at 12 weeks on women with HSIL, showed 14/22 (63.6%, 95%CI: 40.6-82.8%) had no dysplasia and 4/22 (18.2%, 95%CI: 9.9-65.1%) were now low grade demonstrating a combined positive response in 81.8% of women of which 77.8% was confirmed by histology. These data are supported by colposcopic images, which show regression of cervical lesions. CONCLUSIONS: These results demonstrate the potential of Lopimune as a self-applied therapy for HPV infection and related cervical lesions. Since there were no serious adverse events or detectable post-treatment morbidity, this study indicates that further trials are clearly justified to define optimal regimes and the overall benefit of this therapy. TRIAL REGISTRATION: ISRCTN Registry 48776874.

An overview of early investigational drugs for the treatment of human papilloma virus infection and associated dysplasia.

INTRODUCTION: High-risk HPV (HR-HPV) related invasive cervical cancer (ICC) causes >270,000 deaths per annum world-wide with over 85% of these occurring in low-resource countries. Ablative and excisional treatment modalities are restricted for use with high-grade pre-cancerous cervical disease with HPV infection and low-grade dysplasia mostly managed by a watch-and-wait policy. AREAS COVERED: Various pharmacological approaches have been investigated as non-destructive alternatives for the treatment of HR-HPV infection and associated dysplasia. These are discussed dealing with efficacy, ease-of-use (physician or self-applied), systemic or locally applied, side-effects, cost and risks. The main focus is the perceived impact on current clinical practice of a self-applied, effective and safe pharmacological anti-HPV treatment. EXPERT OPINION: Current prophylactic HPV vaccines are expensive, HPV type restricted and have little effect in already infected women. Therapeutic vaccines are under development but are also HPV type-restricted. At present, the developed nations use national cytology screening and surgical procedures to treat only women identified with HPV-related high-grade dysplastic disease. However, since HPV testing is rapidly replacing cytology as the test-of-choice, a suitable topically-applied and low-cost antiviral treatment could be an ideal solution for treatment of HPV infection per se with test-of-cure carried out by repeat HPV testing. Cytology would only then be necessary for women who remained HPV positive. Although of significant benefit in the developed countries, combining such a treatment with self-sampled HPV testing could revolutionise the management of this disease in the developing world which lack both the infrastructure and resources to establish national cytology screening programs.

High-sensitivity human papilloma virus genotyping reveals near universal positivity in anal squamous cell carcinoma: different implications for vaccine prevention and prognosis.

BACKGROUND: Characterisation of human papilloma virus (HPV) infection in anal squamous cell carcinoma (ASCC) may have dual importance: first, aetiological; second, prognostic, informing outcome after chemo-radiotherapy (CRT). We undertook HPV genotyping, and allelic characterisations, to evaluate the aetiological role of HPV while simultaneously evaluating the impact of HPV genotyping on relapse-free (RFS) and overall survival (OS). METHOD: Dual-primer HPV genotyping (subtypes 6, 11, 16, 18, 31, 33, 45, 52, 58) and DNA sequencing of HPV 16 positive tumours were analysed in 151 consecutively referred ASCCs, previously characterised by immunohistochemistry for p16 expression. In 110 patients treated with CRT, factors influencing RFS and OS were evaluated using univariate and multivariate models. RESULTS: HPV positivity was observed in 95%. HPV 16 accounted for 89%; of these, 64% harboured the T350G E6 variant. HPV 16 positivity was significantly correlated with improved 5-year RFS (62% versus 40%; p = 0.027) and OS (59% versus 38%; p = 0.019). p16 expression was also significantly correlated with improved 5-year RFS (positive versus negative: 65% versus 16%; p < 0.0001) and OS (63% versus 13%; p < 0.0001). In multivariable models that included HPV 16 status, p16 status, sex, and age, p16 expression remained an independent prognostic factor for RFS (p < 0.0001) and OS (p = 0.002). CONCLUSION: In ASCC, near-universal HPV detection rates were demonstrated, higher than generally reported in the literature, and supporting the development of multivalent HPV vaccinations for prevention. By contrast, p16 negatively, but not HPV 16 genotype, is an independent adverse prognosticator after chemo-radiotherapy in patients with ASCC.

The PDZ protein Tip-1 is a gain of function target of the HPV16 E6 oncoprotein.

Previous work has indicated that the PDZ domain Tax interacting protein 1 (Tip-1) is a target of the HTLV1 Tax protein and is a potential RhoA effector. We have used the yeast two-hybrid system to show that Tip-1 also interacts with the HPV16 E6 protein. This interaction was confirmed by co-immunoprecipitation from E6 expressing C33A cervical carcinoma cells (C33A-E6) which showed that Tip-1 was not degraded by interaction with the HPV16 E6 oncoprotein. During routine passage we observed that C33A-E6 had a less compact morphology and were less adherent than control vector transfected cells C33A-V cells - a known effect of GTP-RhoA. Comparison of C33A-E6 to C33A-V demonstrated that E6 expressing cells had higher levels of phosphorylated myosin light chains (MLC) and increased cell motility, which was inhibited by antisense silencing of Tip-1 expression and by the RhoA kinase (ROCK) inhibitor Y27632. Both C33A-E6 and C33A-V cells were shown to express GTP activated RhoA. Since ROCKs can be activated by GTP RhoA these data indicate that E6 may increase cell motility by augmenting GTP RhoA mediated activation of ROCKs and that this is dependent on the expression of the Tip-1 protein.

Using HIV drugs to target human papilloma virus.

Over the past decade it has been demonstrated that HIV protease inhibitors have various off-target activities that has enabled them to be repositioned as treatments for a range of other pathologies. Human papilloma virus and related malignancies have been shown to be susceptible to these agents and current progress with this indication is summarized here together with a discussion of the rationale for the off-target effects of these compounds.