Expression Of Human Papillomavirus Type Research Articles

Precancers of the oral mucosa: clinic, diagnostics

The aim of the study. Improving the efficiency of diagnosis and detailing the features of the clinic of «potentially malignant» diseases of the oral mucosa. Clinical and laboratory examination of 124 patients of the department of oral mucosa diseases aged 35 to 80 years, among whom there were 75 women and 49 men, with diseases such as erythroplakia - 12 patients, verrucous leukoplakia - 52 patients, erosive form of leukoplakia - 35 patients, cheilitis Manganotti - 25 patients. Histological and immunohistochemical methods of investigation were used as diagnostics. To assess the proliferative activity of epithelial cells, the determination of the Ki-67 index was used. The synthesis of keratin 15 (K15) in epithelial layers was determined as a diagnostic criterion for the severity of neoplasia. The expression of human papillomavirus type 16 (HPV 16) antigens and p16INK4a protein in epithelial cells was studied, as well as the expression of p53 protein. A high prevalence of p53 mutations was observed in patients with erythroplakia. In leukoplakia, the expression of the Ki-67 protein was detected in the cell nuclei in both the basal and parabasal layers of the multilayer squamous epithelium, in 77% of cases, the expression of the p16INK4a protein in the epithelial nuclei with varying degrees of dysplastic changes was noted, and a positive reaction to HPV16 was also observed in the cell nuclei and cytoplasm of epithelial cells in the basal, parabasal and spiny epithelial layers. The appearance of K15 in the cytoplasm of cells above the basal layer with abrasive precancerous cheilitis was found in 48% of cases. To diagnose early manifestations of neoplastic processes in «potentially malignant» diseases of the oral mucosa, it is necessary to use both classical histological and immunohistochemical methods of investigation with various markers.

Integration and viral oncogene expression of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma and gastric cancer.

Persistent high-risk human papillomavirus (HR-HPV) infections cause cervical cancer and a fraction of head and neck cancer. To investigate whether HR-HPV infection might be also involved in the development of gastric cancer (GC), we developed a platform utilizing a rolling circle amplification (RCA)-based nested L1 polymerase chain reactionwith Sanger sequencing to genotype the HPV DNA in cancer tissues of 361 GC and 89 oropharyngeal squamous cell carcinomas (OPSCC). HPV transcriptional activity was determined by E6/E7 mRNA expression and a 3' rapid amplification of cDNA ends was performed to identify HPV integration and expression of virus-host fusion transcripts. Ten of 361 GC, 2of 89 OPSCC, and 1of 22 normal adjacent tissues were HPV L1 DNA-positive. Five of the 10 HPV-positive GC were genotyped as HPV16 by sequencing and 1of 2 GC with RCA/nested HPV16 E6/E7 DNA detection exhibited HPV16 E6/E7 mRNA. Two OPSCC displayed HPV16 L1 DNA and E6/E7 mRNA, of which 1OPSCC tissue showed virus-host RNA fusion transcripts from an intron region of KIAA0825 gene. Together, our data reveal viral oncogene expression and/or integration in GC and OPSCC and a possible etiology role of HPV infections in gastric carcinogenesis.

HPV-16 Expression and Loss of Cell Differentiation in Primary Bladder Tumors.

Objective Primary bladder tumors have a high degree of malignancy. To investigate the expression of human papillomavirus type 16 (HPV-16) in primary bladder tumors and the loss of cell differentiation and to explore the significance of HPV-16 detection, it is expected to be a disease. Treatment provides a theoretical basis. Methods Fifty-seven patients with primary bladder tumors admitted to our hospital from January 2019 to January 2022 were selected as the research subjects, and they were divided into HPV-related groups according to the human papillomavirus (HPV) infection status (n = 28) and HPV unrelated group (n = 29). The general data of patients were collected, the expression of HPV-16 in bladder tissue samples was detected, and the correlation between pathological parameters and HPV-16 expression was analyzed. Results Among HPV subtypes, HPV 16 subtype accounted for the highest proportion, followed by HPV-18 and HPV-6 subtypes; there was no significant difference in tumor stage (stage 1, stage a, stage 2a) between the HPV-related group and the HPV-unrelated group (stage 1, stage a, and stage 2a). P > 0.05); there was no significant difference in postoperative pathological expression (high expression and low expression) of patients (P > 0.05); there was no statistical difference in age and gender between HPV-related and HPV-unrelated groups (P > 0.05), HPV-related group and HPV-unrelated group compared daily regular drinking and smoking status, the difference was statistically significant (P < 0.05); HPV-16 expression was not correlated with tumor differentiation degree and age of patients (P > 0.05); the area under the curve (AUC) of HPV-16 for judging primary bladder tumor expression and cellular molecular deletion was 0.891, with a sensitivity of 83.94% and a specificity of 88.57%. Conclusion HPV-16 is an upper, expressed in primary bladder tumors and will participate in the differentiation and loss of cells, which can provide effective guidance and basis for the diagnosis of primary bladder tumors, which is an important factor for judging the pathological stage and prognosis of patients and can provide a theoretical reference for the formulation of therapeutic measures.

Expression of Human papillomavirus type 52 L1 capsid gene in Oryza sativa involved in cytoprotective activities

Female cervical cancer is largely formed by Human papillomavirus (HPV), the second leading cause of cancer deaths in women worldwide. HPV-52 is a regionally common high-risk type of cervical cancer found mostly in Asia and reveals geographical variations, in order of importance, as types HPV-16 and -18. However, the differing propensities of HPV types in progressing to cancer, focusing on HPV-52 vaccines, are limited. Several plant-based vaccines against cancer have been developed, and the production of candidate HPV therapeutic vaccines using plant-derived expression platforms is also proven. The objectives of this study were to assess the HPV-52L1 Capsid gene by transferring HPV-52L1 Capsid cDNA into rice (Oryza sativa L.) via an Agrobacterium-mediated transformation, and accumulating HPV-52L1 Capsid proteins in a plant-based expression system to maintain and improve antigenicity. Crude protein extracts containing 5~20 μg from OsHP-52L1 transgenic lines induced cell death and significantly reduced cell proliferation in HPV-positive HeLa cervical cancer cells compared with those non-transformant (NT) rice plants. However, no significant cytotoxicity of induced human breast MDA-MB-231 cell proliferation (as negative control) was observed at any dose compared with NT groups. HeLa cells ameliorated the effects of OsHPV crude protein extracts on cell viability as the extract concentration increased, and treatment with 20 μg of the extract from OsHPV-3 significantly reduced cell viability in HeLa cells (26%) compared with the control group (57%). Our results can be used for exploring the potential of plants for increasing the immunogenicity of OsHPV-52L1 Capsid DNA vaccines, and support the development of cost-effective HPV vaccines, which is highly desirable for resource-poor countries.

A study to assess expression of human papillomavirus types 16 and 18 in oral squamous cell carcinoma using polymerase chain reaction.

Objective:The diverse subset of oral squamous cell carcinoma (OSCC) with different clinical appearance and outcome, independent of traditional risk factors has led to increasing attention in human papillomavirus (HPV) infection.Materials and Methods:The investigation followed a case–control design. Information pertaining to the subjects was retrieved from hospital records. Twenty cases of OSCC and twenty age-matched controls were analyzed to ascertain the prevalence of HPV types 16 and 18. DNA was extracted from the blocks of formalin-fixed paraffin embedded tissues, and HPV-DNA was amplified using HPV type-specific primers by polymerase chain reaction (PCR) method. Data analysis was carried out using Chi-square test.Results:HPV-DNA was detected in 55% of cases (11/20; HPV 16 = 6, HPV 18 = 3 and HPV 16 and 18 = 2) and 30% of controls (6/20; HPV 16 = 3, HPV 18 = 1 and HPV 16 and 18 = 2) indicating higher percentage of HPV presence among OSCC cases. No significant association was found between the presence of HPV and gender, age, site and grade of differentiation of OSCC.Conclusion:Although the presence of HPV was higher in cases compared to controls, none of these differences were statistically significant. HPV 16 and 18 are commonly found in normal oral mucosa mandating the need for distinguishing clinical, subclinical and latent HPV infections.

Langerhans cell homeostasis and activation is altered in hyperplastic human papillomavirus type 16 E7 expressing epidermis.

It has previously been shown that expression of human papillomavirus type 16 (HPV) E7 in epidermis causes hyperplasia and chronic inflammation, characteristics of pre-malignant lesions. Importantly, E7-expressing epidermis is strongly immune suppressed and is not rejected when transplanted onto immune competent mice. Professional antigen presenting cells are considered essential for initiation of the adaptive immune response that results in graft rejection. Langerhans cells (LC) are the only antigen presenting cells located in normal epidermis and altered phenotype and function of these cells may contribute to the immune suppressive microenvironment. Here, we show that LC are atypically activated as a direct result of E7 expression in the epidermis, and independent of the presence of lymphocytes. The number of LC was significantly increased and the LC are functionally impaired, both in migration and in antigen uptake. However when the LC were extracted from K14E7 skin and matured in vitro they were functionally competent to present and cross-present antigen, and to activate T cells. The ability of the LC to present and cross-present antigen following maturation supports retention of full functional capacity when removed from the hyperplastic skin microenvironment. As such, opportunities are afforded for the development of therapies to restore normal LC function in hyperplastic skin.

Human Papillomavirus E7 Oncoprotein Transgenic Skin Develops an Enhanced Inflammatory Response to 2,4-Dinitrochlorobenzene by an Arginase-1-Dependent Mechanism

We have shown that expression of Human Papillomavirus type 16 E7 (HPV16.E7) protein within epithelial cells, as occurs in HPV associated-premalignancy and cancers, results in local immune suppression, and a weak and ineffective immune response to E7 protein. However, a robust acute inflammatory stimulus can overcome this to enable immune elimination of HPV16.E7 transformed epithelial cells. 2,4-Dinitrochlorobenzene (DNCB) can elicit acute inflammation and has been shown to initiate the regression of HPV-associated genital warts. Although clinical use of DNCB is discouraged due to its mutagenic potential, understanding how DNCB induced acute inflammation alters local HPV16.E7 mediated-immune suppression might lead to better treatments. Here, we show that topical DNCB application to skin expressing HPV16.E7 as a transgene induces a hyperinflammatory response, not seen in non-transgenic control animals. The E7 associated-inflammatory response is characterized by enhanced expression of Th2 cytokines and increased infiltration of CD11b+Gr1intF4/80+Ly6ChiLy6Glow myeloid cells, producing arginase-1. Inhibition of arginase with an arginase specific inhibitor, N(omega)-hydroxy-nor-L-arginine, ameliorates the DNCB-induced inflammatory response. Our results demonstrate that HPV16.E7 protein enhances DNCB associated-production of arginase-1 by myeloid cells and consequent inflammatory cellular infiltration of skin.

Differences in the expression of human papillomavirus type 16 (HPV-16) E6 oncogene mRNA in SiHa cell line inoculated with CMV, HSV or ureaplasmas.

One of the factors associated with an increased risk of HPV-related malignant transformation may be bacterial and/or viral infections. The aim of our study was to examine whether the presence of infectious agents commonly detected in the genitourinary tract such as herpesviruses (HSV, CMV), and ureaplasmas (Ureaplasma urealyticum, Ureaplasma parvum) may lead to alterations in the expression of the HPV-16 E6 oncogene. Quantitative RT-PCR analysis was used to assess the level of HPV-16 E6 mRNA expression in SiHa cells. The presence of HSV-1 or HSV-2 in SiHa cells caused a 1.5-fold increase in HPV-16 E6 mRNA expression as compared with non-inoculated SiHa cells. Ureaplasma urealyticum presence but not Ureaplasma parvum stimulated the expression of HPV-16 E6 resulting in a nearly five-fold (4.8) up-regulated E6 mRNA level in SiHa cells. Our study is the first to suggest that infection of Ureaplasma urealyticum in an urogenital tract could increase the risk of cervical cancer by overexpression of the HPV E6 oncogene.

Pericentromeric Regions Are Refractory To Prompt Repair after Replication Stress-Induced Breakage in HPV16 E6E7-Expressing Epithelial Cells

Chromosomal instability is the major form of genomic instability in cancer cells. Amongst various forms of chromosomal instability, pericentromeric or centromeric instability remains particularly poorly understood. In the present study, we found that pericentromeric instability, evidenced by dynamic formation of pericentromeric or centromeric rearrangements, breaks, deletions or iso-chromosomes, was a general phenomenon in human cells immortalized by expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7). In particular, for the first time, we surprisingly found a dramatic increase in the proportion of pericentromeric chromosomal aberrations relative to total aberrations in HPV16 E6E7-expressing cells 72 h after release from aphidicolin (APH)-induced replication stress, with pericentromeric chromosomal aberrations becoming the predominant type of structural aberrations (∼70% of total aberrations). In contrast, pericentromeric aberrations accounted for only about 20% of total aberrations in cells at the end of APH treatment. This increase in relative proportion of pericentromeric aberrations after release from APH treatment revealed that pericentromeric breaks induced by replication stress are refractory to prompt repair in HPV16 E6E7-expressing epithelial cells. Telomerase-immortalized epithelial cells without HPV16 E6E7 expression did not exhibit such preferential pericentromeric instability after release from APH treatment. Cancer development is often associated with replication stress. Since HPV16 E6 and E7 inactivate p53 and Rb, and p53 and Rb pathway defects are common in cancer, our finding that pericentromeric regions are refractory to prompt repair after replication stress-induced breakage in HPV16 E6E7-expressing cells may shed light on mechanism of general pericentromeric instability in cancer.

Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants

Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108-120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2 108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2 108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2 108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2 108-120 epitope were found after both methods of vaccine delivery.

The expression of human papillomavirus type 16 (HPV16�E7) induces cell cycle arrest and apoptosis in radiation and hypoxia resistant glioblastoma cells

p53 is a widely known tumor-suppressor gene product that plays a key role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents. Human glioma cells with functional p53 are known to be more resistant to γ-radiation. The aim of this study was to investigate whether the mutant glioblastoma cells (U87MG) transfected with human papilloma virus-type 16 E7 (HPV16 E7) genes were capable of increasing sensitivity towards irradiation and hypoxia-induced cell death. The pLXSN retroviral vector expressed HPV-16E7 genes and was infected into the p53 mutated U87MG cell line. A specific amplification band of HPV16 E7 genes was detected in E7 genes and transfected in the U87MG cell line using a reverse transcriptase polymerase chain reaction. The experimental groups included the mutant glioblastoma cell line (U87MG), empty vector (pLXSN) transfected to mutant glioblastoma cell line (U87MG-LXSN), and retrovirus carrying HPV16 E7 genes transfected to the mutant glioblastoma cell line (U87MG-E7). Hypoxic conditions were optimized using LDH assay and the subjects were exposed to hypoxia (16 and 20 h) and irradiation (9 h). Hoechst-propidium iodide (PI) staining results showed that hypoxia and irradiation increased the number of dead cells in the U87MG-E7 cells compared to U87MG and U87MG-LXSN cells. Results of the FACS analysis showed a similar pattern and recorded 80.44 and 58.94% of apoptotic cells in U87MG-E7 and U87MG cells, respectively. Cell cycle analysis by FACS revealed that, following irradiation and hypoxia, cells showed G2-M arrest. Additionally, the Western blot analysis results showed altered expression of E2F-1, Rb and p53 in the irradiation- and hypoxia-induced U87MG-E7 cells compared to U87MG and U87MG LXSN cells. In conclusion, the over-expression of HPV16 E7 genes in U87MG cell lines increasd cell apoptosis and E2F1 expression compared to the HPV non-infected U87MG cells following irradiation and hypoxia. These results indicate that tumor-specific therapies that increase sensitivity towards radiation and hypoxic conditions may be beneficial for suppression of cancers.

Transcriptome Sequencing Demonstrates that Human Papillomavirus Is Not Active in Cutaneous Squamous Cell Carcinoma

Beta-papillomavirus (β-HPV) DNA is present in some cutaneous squamous cell carcinomas (cuSCC), but no mechanism of carcinogenesis has been determined. We used ultra-high throughput sequencing of the cancer transcriptome to assess whether papillomavirus transcripts are present in these cancers. Sixty-seven cuSCC samples were assayed for β-HPV DNA by PCR, and viral loads were measured with type-specific qPCR. Thirty-one SCCs were selected for whole transcriptome sequencing. Transcriptome libraries were prepared in parallel from the HPV18 positive HeLa cervical cancer cell line and HPV16 positive primary cervical and periungual SCC. Thirty percent (20/67) of the tumors were positive for β-HPV DNA, but there was no difference in β-HPV viral load between tumor and normal tissue (p=0.310). Immunosuppression and age were significantly associated with higher viral load (p=0.016 for immunosuppression; p=0.0004 for age). Transcriptome sequencing failed to identify papillomavirus expression in any of the skin tumors. In contrast, HPV 16 and 18 mRNA transcripts were readily identified in primary cervical and periungual cancers and HeLa cells. These data demonstrate that papillomavirus mRNA expression is not a factor in the maintenance of cuSCC.

Isolation and Characterization of an RNA Aptamer for the HPV-16 E7 Oncoprotein

Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer.

Expression of human papillomavirus type 16 (HPV16) L1 protein in Pichia pastoris

To investigate the production of human papillomavirus type 16 (HPV16) L1 structural protein in Pichia pastoris , the effect of different gene constructs and varying cultivation conditions on production levels were assessed. The highest volumetric production levels in P. pastoris were obtained with a human codon-optimised gene construct. This construct displayed both the lowest codon adaptation index (CAI) and the highest guanine-cytosine (GC) content of the three constructs investigated. Dissolved oxygen (DO) controlled cultivations produced superior results compared to a constant methanol feeding (CMF) strategy, delivering the highest volumetric HPV L1 protein concentration (104.8 µg.l -1 ) at a 20% DO set-point. This was concomitant with an increased biomass concentration in the DO controlled cultivations.

Expression of human papillomavirus type 16 E7 is sufficient to significantly increase expression of angiogenic factors but is not sufficient to induce endothelial cell migration

Tumor suppressors negatively regulate angiogenesis, an essential step in tumor progression. Together, HPV 16 E6 and E7 proteins, which target p53 and pRb family members, respectively, for degradation, increase the expression of two angiogenic inducers, VEGF and IL-8, in primary foreskin keratinocytes (HFKs). Conditioned media from such cells are sufficient to alter endothelial cell behavior. Here, the individual contribution of E6 and E7 to angiogenesis was investigated. E7 and, to a lesser extent E6, increased expression of VEGF and IL-8. Nevertheless, neither conditioned media from HPV 16 E6 nor E7-expressing HFKs were sufficient to induce migration of endothelial cells. Conditioned media from HFKs expressing the HPV 16 E6 and the E7 mutant E7C24G, which can target p107 and p130 but not pRb for degradation, contained increased levels of VEGF and IL-8. The results suggest that the mechanism of HPV 16 E7-mediated increased levels of VEGF is pRb-independent.

Cystatin C Deficiency Promotes Epidermal Dysplasia in K14-HPV16 Transgenic Mice

BackgroundCysteine protease cathepsins are important in extracellular matrix protein degradation, cell apoptosis, and angiogenesis. Mice lacking cathepsins are protected from tumor progression in several animal models, suggesting that the regulation of cathepsin activities controls the growth of various malignant tumors.Methods and ResultsWe tested the role of cathepsins using a mouse model of multistage epithelial carcinogenesis, in which the human keratin-14 promoter/enhancer drove the expression of human papillomavirus type 16 (HPV16) early region E6/E7 transgenes. During the progression of premalignant dysplasia, we observed increased expression of cysteine protease cathepsin S, but concomitantly reduced expression of cathepsin endogenous inhibitor cystatin C in the skin tissue extract. Absence of cystatin C in these transgenic mice resulted in more progression of dysplasia to carcinoma in situ on the face, ear, chest, and tail. Chest and ear skin extract real time PCR and immunoblot analysis, mouse serum sample ELISA, tissue immunohistological analysis, and tissue extract-mediated in vitro elastinolysis and collagenolysis assays demonstrated that cystatin C deficiency significantly increased cathepsin expression and activity. In skin from both the chest and ear, we found that the absence of cystatin C reduced epithelial cell apoptosis but increased proliferation. From the same tissue preparations, we detected significantly higher levels of pro-angiogenic laminin 5-derived γ2 peptides and concurrently increased neovascularization in cystatin C-deficient mice, compared to those from wild-type control mice.ConclusionEnhanced cathepsin expression and activity in cystatin C-deficient mice contributed to the progression of dysplasia by altering premalignant tissue epithelial proliferation, apoptosis, and neovascularization.

Expression of human papillomavirus type 16/18 in human cervical carcinomas by the quantum dot fluorescent in-situ hybridization

To investigate fluorescence in situ hybridization labeled with quantum dots (QDs) for the detection of human papillomavirus 16/18 (HPV16/18) infection in cervical carcinoma patients. A total of 80 biopsy samples of squamous carcinoma of cervix were assayed for HPV 16/18 infection by using quantum dot labeled fluorescent in situ hybridization (QD-FISH) and chromogenic in situ hybridization (CISH) techniques, respectively. The results obtained by using two different methods were statistically analyzed. The positive rate for HPV16/18 by QD-FISH was 88.8% (71/80), higher than that (80.0%) by CISH, however, the result was statistically not significant (P=0.127). The positive detection rates for HPV16/18 by using both methods increased coincidentally with raising of the tumor grading stage. The sensitivity and specificity of HPV infection detectable by QD-FISH is higher than that by the CISH technique.

MicroRNA 203 Expression in Keratinocytes Is Dependent on Regulation of p53 Levels by E6

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

Transient expression of human papillomavirus type 16 virus-like particles in tobacco and tomato using a tobacco rattle virus expression vector

The major capsid protein L1 of human papillomavirus type 16 (HPV16) was transiently expressed in tobacco (Nicotiana benthamiana and Nicotiana tabacum) and tomato (Lycopersicon esculentum) leaves using Agrobacterium tumefaciens. The expression vector pTV00 was derived from tobacco rattle virus (TRV). The highest L1 expression 15 μg g-1(f.m.) was achieved when the coding sequence of L1 was optimized for expression in humans that caused an increase of the guanine and cytosine (GC) content from 38.2 % in wild type HPV16 to 64.1 % in optimized sequence. L1 monomers readily self-assembled into capsomeres and further into virus like particles (VLPs). Immunological characterization and electron microscopy showed that 89 % of L1 retained VLP structure also in extracts prepared from freeze-dried leaves. Plant expressed L1 in crude extracts was highly immunogenic without any additional adjuvant as vaccinated mice developed strong humoral and cellular immune response, comparable to that elicited by purified VLPs derived from insect cells. Further, the induced antibodies effectively neutralized infection of 293TT cells with pseudovirions. This finding demonstrates that the TRV expression system is comparable to other plant expression systems and due to the broad host range of TRV is particularly attractive when expression in plants with low content of toxic alkaloids is desired. Moreover, a monoclonal anti-L1 antibody E2 raised in the course of immunization with crude extract from freeze-dried leaves expressing L1 is specific preferentially against HPV VLPs and could be used in direct ELISA for monitoring of VLPs assembly and VLP purification protocols.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-α

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12;16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPV-associated disease development is warranted.