OBJECTIVEHuman papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study.

METHODSThe whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins.

RESULTSThree clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography.

CONCLUSIONHPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.

Capsid Proteins ; biosynthesis ; genetics ; isolation & purification ; Cloning, Molecular ; Genes ; Papillomaviridae ; genetics ; Pichia ; genetics ; metabolism ; Viral Proteins

OBJECTIVETo evaluate the correlation of the expression of homeodomain-interacting protein kinase 2 (HIPK2) with human papillomavirus (HPV) infection and apoptosis in cervical cancer.

METHODSFormalin-fixed, paraffin embedded tissue samples from 50 cervical cancers and 15 normal uterine cervix cases were obtained. Apoptosis was quantified by TdT-mediated dUTP nick end labeling (TUNEL) assay and the expression of HIPK2 as well as HPV by immunohistochemical staining.

RESULTSHIPK2 protein expression was detected in 88.0% (44/50) of cervical cancers and 6.7% (1/15) of normal cervical tissues. HPV was found in 78.0% (39/50) of cervical cancers and 20.0% (3/15) of normal cervical tissue samples. The expression of HIPK2 protein was significantly and positively correlated with HPV presence (r=0.467, P<0.01), but negatively with apoptotic index (r=-0.370, P<0.05).

CONCLUSIONHIPK2 protein expression is positively correlated with HPV infection, but negatively with apoptotic index in cervical cancers. Therefore, HIPK2 may be involved in the mechanism of apoptosis in cervical cancer and may play an important role in cervical carcinogenesis.

Adenocarcinoma ; metabolism ; pathology ; virology ; Apoptosis ; Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Carrier Proteins ; metabolism ; Cervix Uteri ; metabolism ; Female ; Humans ; Middle Aged ; Papillomaviridae ; Papillomavirus Infections ; Proliferating Cell Nuclear Antigen ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology

OBJECTIVETo perform an comparative proteome analysis of human papillomavirus-infected cervical specimens and to investigate different expressions between high- and low-risk genotypes.

METHODSThe cervical specimens were divided into two groups (cervical intraepithelial neoplasia group and condyloma acuminatum group) according to their genotypes. Using comparative proteome technology, high-risk human papillomavirus-infected cervical intraepithelial neoplasia, low-risk human papillomavirus-infected condyloma acuminatum, and normal cervical intraepithelial tissue were compared. The differential expression protein spots were identified by mass spectrometry.

RESULTSTotally 26 differential spots were selected and analyzed, and 22 peptide mass fingerprints (PMF) maps were obtained by MALDI-TOF-MS. Eighteen proteins were preliminarily identified after searching the NCBInr database. The function information of these 18 proteins mainly involved cell metabolism, signal transduction, cell secretion, cell cytoskeleton construction, cell proliferation, and apoptosis.

CONCLUSIONThe proteomic expressions after the cervical infection of high- or low-risk genotype of human papillomavirus are obviously different.

Cervical Intraepithelial Neoplasia ; metabolism ; virology ; Cervix Uteri ; metabolism ; Condylomata Acuminata ; metabolism ; virology ; Female ; Genotype ; Humans ; Papillomaviridae ; genetics ; pathogenicity ; Papillomavirus Infections ; metabolism ; virology ; Proteome ; metabolism ; Risk ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Uterine Cervical Diseases ; metabolism ; virology

OBJECTIVETo study the correlation of the expressions of CD82 and hTERT and HPV infection with the clinical pathological features of penile cancer and identify their prognostic significance in the lymphatic metastasis of the disease.

METHODSA total of 44 patients underwent partial or radical penectomy and lymph node dissection. The expressions of CD82 and hTERT were determined by immunohistochemistry, and HPV infection was detected by PCR.

RESULTSThe positive rates of CD82, hTERT, and HPV DNA in penile carcinoma were 47.7%, 38.6% and 25.9%, respectively. The amplified HPV DNA was HPV-16. The pathological stage and hTERT expression were positively correlated with inguinal lymph node metastasis of penile cancer (P = 0.032, P = 0.041), and so was the pathological stage with the expression of CD82 (P = 0.045), but neither the pathological stage, nor the expression of CD82 or the positive rate of HPV DNA showed any correlation with lymph node metastasis (P = 0.627, P = 0.094, P = 0.633).

CONCLUSIONThe pathological grade and hTERT expression are independent prognostic factors for lymph node metastasis in penile carcinoma. These features help the prognosis and identification of the patient at the risk of nodal metastasis.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Humans ; Kangai-1 Protein ; metabolism ; Male ; Middle Aged ; Neoplasm Staging ; Papillomaviridae ; Papillomavirus Infections ; metabolism ; Penile Neoplasms ; metabolism ; pathology ; virology ; Telomerase ; metabolism

OBJECTIVETo define a correlation between different human papillomavirus (HPV) types and telomerase activity in cervical cancer.

METHODSTelomerase activity was detected by TRAP-PCR, and different HPV type was determined by PCR in 83 cervical cancer, 47 cervical intraepithelial neoplasia (CIN) and 10 normal cervix cases.

RESULTSWith regard to positive rates of telomerase and HPV 16/18: the results were cervical cancer > CIN > normal cervix, CIN III > CIN I, II; with regard to HPV 6/11 positive rate: the results showed CIN I, II > CIN III. Positive rates of telomerase cervical cancer and HPV were bearing on grading and staging, but they did not correlate with histologic subtypes. Positive rate of HPV 6/11 had nothing to do with grading, staging and histologic patterns. On expression strength of telomerase and HPV 16/18: the results showed cervical cancer > CIN, CIN III > CIN I, II. Regard to HPV 6/11'expression strength: the results showed CIN I, II > CIN III, CIN > cervical carcinoma.

CONCLUSIONHPV 16/18 infection seemed to have played an important role in carcinogenesis of cervical lesions by activation of telomerase.

Cervical Intraepithelial Neoplasia ; enzymology ; pathology ; virology ; Female ; Humans ; Neoplasm Staging ; Papillomaviridae ; isolation & purification ; Telomerase ; metabolism ; Uterine Cervical Neoplasms ; enzymology ; pathology ; virology

OBJECTIVETo study the clinical efficacy and mechanisms of Youdujing (YDJ) preparation in treating the cervical high-risk human papilloma virus (HR-HPV) infection.

METHODSTotally HR-HPV infection 70 patients were assigned to the treatment group and the control group using random single blind method, 35 cases in each group. YDJ external lotion and YDJ cream were applied to patients in the treatment group, while normal saline was applied for those in the control group. HR-HPV DNA detection was performed by the end of the 1st menstruation after 3 menstrual cycles. The cervical biopsy and cervical smear were performed using vaginoscope before and after treatment. The mRNA expression of human telomerase reverse transcriptase (hTERT) was detected from fresh tissues of the cervical lesion.

RESULTSThe total effective rate was 96.6% in the treatment group, higher than that of the control group (70.00%, P<0.01). Before treatment there was no statistical difference in the pathological results of the cervix and the mRNA expression of hTERT between the two groups (P>0.05). The mRNA expression of hTERT decreased in the two groups after treatment, showing statistical difference (P<0.01). Better effects on the pathological results of the cervix and the mRNA expression of hTERT were obtained in the treatment group after treatment (P<0.05).

CONCLUSIONYDJ played a role in clearing HR-HPV infection and reversing the cervical precancerous changes possibly through down-regulating the mRNA expression of hTERT and lowering the telomerase activation.

Adult ; Cervix Uteri ; virology ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Papillomaviridae ; Papillomavirus Infections ; drug therapy ; virology ; Phytotherapy ; RNA, Messenger ; genetics ; Single-Blind Method ; Telomerase ; metabolism ; Treatment Outcome

HPV-2 is a very common type of HPV which causes common warts. The E2 protein of virus can repress the activity of the viral early promoter through binding to the specific binding sites in viral LCR. Previously we reported that the repression of a mutated E2 protein of HPV-2 isolated from a patient with huge common wart on the viral early promoter was obviously decreased, and A338V mutation located at the C terminal DNA binding region of E2 protein. In this study, we expressed and purified the recombinant mutated and prototype E2 fusion proteins, both in the contexts of the C terminal and the full length, by prokaryotic expression system. The electrophoretic mobility shift assay showed E2 protein could bind to double-stranded DNA oligos labeled with biotin that covered two E2 binding sites. The DNA binding abilities of both C terminal and full-length mutated E2 proteins were stronger than the prototype analogs. This result indicates that the enhancement of the mutated E2 DNA binding ability may be the molecular mechanism for its impact on the activity of viral promoter, which correlates with the phenotype of extensive common wart.
DNA ; metabolism ; DNA-Binding Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Electrophoresis ; Genetic Vectors ; genetics ; Mutation ; Papillomaviridae ; Promoter Regions, Genetic ; genetics ; Protein Binding ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Viral Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism

OBJECTIVETo clarify the role of midkine (MK) in vulvar carcinogenesis though examination of its expression in vulvar lesions including vulvar condyloma acuminata (VCA), vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell carcinomas (VSCC), and to analyze the relationship between MK expression and human papilloma virus (HPV) infection.

METHODSThirty VSCC, 15 VIN and 10 VCA patients were studied by streptavidin-biotin-immunoperoxidase method. MK expression was compared with clinicopathologic features of vulvar tumors.

RESULTSMK was expressed in 26 of 30 VSCC (87%), 3 of 5 VIN III and all VCA samples, whereas no MK expression was detected in the VIN I-II samples or in normal epithelium. The difference of MK expression between VIN III and VSCC was statistically significant (P < 0.05). MK was more intensely expressed in differentiated-type (well differentiated and moderately differentiated) VSCC than in undifferentiated-type (poorly differentiated) VSCC. There was no statistically significant correlation between MK expression and clinical stage, lymph node metastasis and HPV infection in VSCC. MK expression were observed in all HPV-positive specimens including 2 VSCC, 1 VIN III and all VCA.

CONCLUSIONSMK gene expression may be a late event in vulvar squamous cell malignant transformation, and may be associated with vulvar tumor cell differentiation. HPV-positive vulvar tumors expressed MK protein.

Carcinoma, Squamous Cell ; chemistry ; virology ; Carrier Proteins ; biosynthesis ; Condylomata Acuminata ; metabolism ; virology ; Cytokines ; Female ; Humans ; Papillomaviridae ; chemistry ; Papillomavirus Infections ; metabolism ; Precancerous Conditions ; chemistry ; virology ; Tumor Virus Infections ; metabolism ; Vulvar Diseases ; metabolism ; virology ; Vulvar Neoplasms ; chemistry ; virology

BACKGROUND: Head and neck cancers (HNCs) are a heterogeneous group of tumors that progress owing to varied enviromental and genetic risk factors. Viral infections are threatening and adept at altering the expression of cellular transcription factors such as nuclear factor kappa B (NF-κB) and deregulation of other cellular proteins like NF kappa B inhibitor alpha (IκBα). The present study was conducted to detect high-risk genotypes of human papillomavirus (HPV) and protein expression of NF-κB signaling pathway in HNC patients with HPV infection. METHODS: For HPV detection, genomic DNA from 152 HNC tumors was extracted formalin-fixed paraffin-embedded tissue DNA kit. For genotyping, polymerase chain reaction (PCR) using a general primer, HPV type-specific primers and agarose gel electrophoresis were performed. Immunohistochemistry (IHC) was also performed on 4-μm thick tissue sections using HPV E6 monoclonal antibody. Protein expression analysis of NF-κB signaling pathway including p50, p65, and IκBα was performed using IHC. RESULTS: PCR analysis showed that 24.3% (37/152) of HNC cases were HPV positive. Among HPV positive, 86.5% (32/37) were tobacco users, while among HPV negative, 66.9% (77/115) were tobacco users. A significant association of HPV positivity and tobacco user was observed by univariate analysis [ P   <  0.01; odds ratio (OR): 0.310, 95% confidence interval (CI): 0.110 to 0.870]. More HPV positive patients were with poor oral hygiene (78.3%) when compared with patients with good oral hygiene (21.6%) [ P  < 0.03, OR: 2.440, 95% CI: 1.650 to 3.600]. The results of the logistic regression analysis showed that age, tobacco use and oral hygiene are significant predictors ( P  < 0.02). PCR and IHC staining results confirmed that HPV16 was predominant among HNC cases (64.8%) when compared with HPV18 (35.2%). Expression of NF-κB proteins (p50, p65, and IκBα inhibitor) were also observed in HPV and non-HPV infected HNC tissues. IHC expression of p50, and p65 showed nuclear staining, while IκBα inhibitor showed cytoplasmic staining. Protein expression in HPV cases was higher as compared to HPV naive cases ( P  < 0.05). CONCLUSIONS From the study, it can be established that the use of tobacco, oral hygiene, and HPV infection may be synergistically involved in modulating the expression of NF-κB signaling pathway for the development and progression of HNC in the Pakistani population.
Alphapapillomavirus ; Antibodies, Monoclonal ; DNA ; DNA, Viral/genetics* ; Formaldehyde ; Head and Neck Neoplasms ; Humans ; NF-KappaB Inhibitor alpha/genetics* ; NF-kappa B/metabolism* ; Oral Hygiene ; Pakistan ; Papillomaviridae/metabolism* ; Papillomavirus Infections/metabolism* ; Signal Transduction ; Tobacco ; Tobacco Use ; Transcription Factors/metabolism*

OBJECTIVETo construct the prokaryotic and eukaryotic expression vectors pET32/E5 and pcDNA3.1/E5 for transformation into E. coli BL21 and NIH(3)T(3) cells respectively to observe the expression of human papillomavirus type 16 E5 protein (HPV16 E5).

METHODSHPV16 E5 gene was amplified by PCR from clinical isolates of HPV 16 and inserted into the plasmid pET32a(+) followed by digestion with BamH I and Hind III. The recombinant plasmid pET32/E5 was transformed into E. coli JM109 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/E5 were verified by restriction endonucleases BamH I and Xho and sequence analysis. The expression of HPV16 E5-TRX fusion protein in E. coli BL21(ED3) was identified by SDS-PAGE and Western blotting. The digestion product of BamH I and Xho was purified and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct pcDNA3.1/E5, which was identified by sequencing and transfected into NIH3T3 cells. The NIH(3)T(3) cells with stable expression of HPV16 E5 were selected by G418 and confirmed by RT-PCR.

RESULTSThe pET32/E5 and pcDNA3.1/E5 vectors were constructed successfully. E.coli BL21(DE3) transformed by the recombinant plasmid pET32/E5 expressed HPV16 E5-TRX fusion protein efficiently. In the presence of 1 mmol/L IPTG at 28 degrees C, HPV16 E5-TRX recombinant protein accounted for about 10% of the total bacterial proteins. NIH3T3 cells stably expressing HPV16 E5 were harvested by selection with 250 g/ml of G418. HPV16 E5 gene from pcDNA3.1/E5-transfected NIH(3)T(3) cells was amplified by RT-PCR, and sequence analysis demonstrated the acquisition of the full-length gene fragment.

CONCLUSIONSThe prokaryotic and eukaryotic vectors for the HPV16 E5 gene have been successfully constructed. The acquisition of E .coli and NIH(3)T(3) cells with stable expression of HPV16 E5 protein may facilitate subsequent research of the biological properties and the transformation mechanism of HPV16 E5 protein on specific cells.

3T3 Cells ; Animals ; Escherichia coli ; genetics ; metabolism ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Humans ; Mice ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomaviridae ; genetics ; isolation & purification ; Papillomavirus Infections ; virology ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics