This study was intended to establish a method of purification of HPV16 L1 protein expressed in a prokaryotic system and to obtain the purified protein. The prokaryotic expression vector pGEX-4T-1-HPV16 L1 was constructed and transformed into E. coli BL21 cell, and induced by 1 mM IPTG to express HPV16L1 protein. The inclusion bodies were isolated and solubilized with 8 M urea. After the urea was removed by gradual dialysis, the denatured L1 protein were renatured and then were purified by affinity chromatography. The results showed that HPV16L1 protein formed inclusion bodies in bacterial expression system, suggesting that this assay can be used to purify HPV16L1 protein and hence provide a basis for studying the applications of HPV16 L1 protein.
Capsid Proteins ; biosynthesis ; Escherichia coli ; metabolism ; Genetic Vectors ; Human papillomavirus 16 ; Oncogene Proteins, Viral ; biosynthesis

OBJECTIVE: To investigate the molecular mechanism of Wnt5a and Epstein-Barr virus latent membrane protein 1 (LMP1) aberrant expression in the nasopharyngeal carcinogenesis and to estimate if it can act as a molecular marker for nasopharyngeal cancer (NPC). METHODS: Immunohistochemistry combined with previously made tissue microarrays were used to study the expression of Wnt5a and LMP1 in the nasopharyngeal carcinogenesis tissues. We investigated the role of over expression of Wnt5a and LMP1 in the development and progression of NPC and their relation with the clinicopathological features of NPC and whether they could act as molecular markers in benign and malignant NPC. RESULTS: The positive percentage of Wnt5a and LMP1 protein expression in the NPC was significantly increased as compared with that in atypically hyperplastic nasopharyngeal epithelium, hyperplastic nasopharyngeal epithelium and histologically normal nasopharyngeal epithelium (P<0.05, P<0.01, and P<0.01). Wnt5a and LMP1 proteins were significantly higher in atypically hyperplastic nasopharyngeal epithelium than those in the hyperplastic nasopharyngeal epithelium and normal nasopharyngeal epithelium (P<0.05 and P<0.01). The positive expression of Wnt5a and LMP1 proteins in clinical T3 and T4 staged NPC was higher than that in clinical T1 and T2 staged NPC (P<0.01 and P<0.05). The positive expression of Wnt5a protein in the NPC with lymph node metastasis was higher than that in the NPC without lymph node metastasis (P<0.01). The positive percentage of LMP1 protein was significantly increased in non-keratinizing carcinoma compared with undifferentiated carcinoma and keratinizing carcinoma (P<0.05 and P<0.05). The expression of Wnt5a protein in the NPC had significant positive correlation with LMP1 (r=0.354, P<0.001). Combined molecular phenotype of both Wnt5a and LMP1 expression was a good marker to distinguish NPC from non-cancerous nasopharyngeal epithelium. CONCLUSION The expression of Wnt5a and LMP1 protein in the NPC is positively correlated, and both wnt5a and LMP1 protein play important roles in the nasopharyngeal carcinogenesis either together or successively promoting the malignant transformation of nasopharyngeal epithelium and the development and progression of NPC. Both Wnt5a and LMP1 positive expression may act as good markers for NPC differential diagnosis.
Biomarkers, Tumor ; genetics ; metabolism ; Carcinogenesis ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Oncogene Proteins, Viral ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Tissue Array Analysis ; Viral Matrix Proteins ; genetics ; metabolism ; Wnt Proteins ; genetics ; metabolism ; Wnt-5a Protein

Human papilloma virus (HPV) is believed to be an essential factor for the development of cervical cancer. Early diagnosis and treatment of cervical intraepithelial neoplasia can effectively inhibit the future progression. HPV late 1 protein possesses epitope that can identify and adhere to host cells, and thus may play an important role in HPV infection and cervical carcinogenesis.
Capsid Proteins ; Cervix Uteri ; metabolism ; virology ; Female ; Humans ; Oncogene Proteins, Viral ; Papillomavirus Infections ; complications ; Uterine Cervical Neoplasms ; virology

BACKGROUNDHeat shock protein 70 (HSP70) is expressed highly in epithelial tumours associated closely with human papillomavirus 16 (HPV16) infections. However, evidence about the direct relationship between HSP70 expression and HPVs infections are still lacking. In the present study, we examined the expression of HSP70 in keratinocytes introduced with HPV16 E6/E7 oncogenes.

METHODSStable transfected cells were established by transfection of the plasmids pLXSN16E6/E7 into cultured primary keratinocytes and subsequently selected by plasmid specific selection antibiotic (G418) at the required concentration. The expression of HSP70 in pLXSN16E6/E7 transfected keratinocytes was determined by Western blot. The correlation of HSP70 expression and E6/E7 transfection was further confirmed by doubly labelled immunofluorescent staining.

RESULTSCompared to non-transfected keratinocytes, there was a significant trend for higher levels of HSP70 in pLXSN16E6/E7 transfected keratinocytes. Doubly labelled immunofluorescent staining experiment showed that the co-localization of HPV16 E6/E7 and HSP70 in transfected keratinocytes was observed and increased expression of HSP70 was strongly associated with the transfection of HPV16 E6/E7.

CONCLUSIONSOur studies demonstrated increased levels of HSP70 proteins in keratinocytes stably transfected by HPV16 E6/E7 oncogenes. It suggests that the expression of HSP70 is modulated by HPV16 E6/E7 proteins, which may be involved in HPV16 E6/E7 induced immortalization.

Cells, Cultured ; HSP72 Heat-Shock Proteins ; biosynthesis ; Humans ; Keratinocytes ; metabolism ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins ; genetics ; Transfection

High-risk human papillomavirus (HPV) is the principal cause of various cancers including cervical cancer, anal cancer, vulvar cancer, and some head and neck cancers. In the viral life cycle, by interacting with both viral and host DNA and proteins, the HPV E2 protein plays a pivotal role in viral transcriptional regulation and DNA replication, and it is also associated with modification of various cellular processes, including host gene transcription, RNA processing, apoptosis, ubiquitination, and intracellular trafficking, to create a convenient environment for a replicative cycle of the virus and contribute to the HPV pathogenesis. Elucidating the roles of E2 protein throughout the viral life cycle will improve our understanding of the viral life cycle and pathogenesis and help us identify novel antiviral agents with therapeutic potential. This article reviews the research progress in the structure, roles, and activity of high-risk HPV E2 protein, particularly that of HPV-16.
Animals ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Gene Expression Regulation, Viral ; Human papillomavirus 16 ; genetics ; metabolism ; Humans ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus Infections ; genetics ; metabolism ; virology ; Uterine Cervical Neoplasms ; genetics ; metabolism ; virology

This study was aimed to identify pET21b-HPV16E2/BL21(DE3) strain and to optimize the expression of human papillomavirus type 16 (HPV16) E2 protein by orthogonal analysis. Four influence factors on two levels were selected to increase the target protein quantity. The four factors were induction time, induction temperature, inductor concentration and cell density. The quantity of HPV16 E2 protein was used as the evaluation parameter. Induced by IPTG, HPV16 E2 protein was analyzed by SDS-PAGE and Western Blot. Target protein was analyzed by GIS imaging system to quantify the protein level. SPSS13. 0 software was applied to analyze the result. Data showed that the expression strain pET211rHPV16 E2/BL21(DE3) was identified correctly. HPV16 E2 protein expressed mainly at insoluble form. The 42KD protein band was identified by SDS-PAGE and Western blot. Orthogonal test was applied on influence factor analysis and expression optimization successfully. Main influence factors were inductor concentration and induction temperature. The optimimum condition of maximum expression quantity was 37 degrees C, 7h, 1.0 mmol/L IPTG and OD600 1.0. In this experiment, orthogonal test could not only be used to analyze the influential factors and promote the target protein expression, but also be used to provide a better experiment method for molecular biological study.
DNA-Binding Proteins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; metabolism ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus Infections ; virology ; Recombinant Proteins ; biosynthesis ; genetics

The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZa-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPVI6L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37 degrees C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.
Capsid Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; genetics ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

The relationship between Human papillomavirus (HPV) 16 infection and the natural course of colorectal adenocarcinoma has not been fully defined. In this study, the HPV 16 E7 DNA was detected in 82 patients with primary colorectal adenocarcinoma to study the relationship between HPV 16 infection and colorectal carcinoma. Samples were taken from both the tumors and the adjacent normal mucosa in the same patient. Polymerase chain reaction (PCR) was used to amplify the HPV16 E7 DNA fragment. The PCR products were gel-purified and sequenced for HPV genotyping. DNA sequence analysis indicated that PCR product was 297 bp. It was the equivalent of 562-858th pairs in the HPV 16 primitive sequences. Our results showed HPV16 E7 DNA expression was significantly higher in colorectal carcinoma (42/82) than in adjacent normal mucosa (4/82). The correlation was found between HPV16 E7 expression and tumor's location; the positive rate was 18.18% in the ascending colon carcinoma group and 64.10% in the rectal carcinoma group. High HPV16 E7 expression was also associated with lower Dukes stage (P < 0.01). These results indicated that there was correlation between colorectal adenocarcinoma and HPV 16 infection. HPV16 infection was relatively higher in the colorectal carcinoma and rare in the adjacent normal mucosa. Specimens expressing higher levels of HPV 16 E7 DNA were associated with lower Dukes stage and more distal location.
Adenocarcinoma ; metabolism ; virology ; Adult ; Aged ; Aged, 80 and over ; Colorectal Neoplasms ; metabolism ; virology ; DNA, Viral ; analysis ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomaviridae ; isolation & purification ; Papillomavirus E7 Proteins ; Papillomavirus Infections ; metabolism ; virology

OBJECTIVE: To investigate the expression of high risk human papilloma virus (HPV) 16-E6 protein in non-neoplastic epithelial disorders of the vulva (NNEDV) and squamous cell carcinoma of the vulva (VSCC), and to explore whether HPV16-E6 protein is the etiological factor in NNEDV and its correlation with squamous cell carcinoma of the vulvae. METHODS: We detected HPV16-E6 protein expression in 15 normal vulvae cases, 40 NNEDV cases and 45 VSCC cases by immunohistochemistry SP method. RESULTS: The positive rate of HPV16-E6 in different vulva tissues: was 0% in the normal vulva, 30% in NNEDV and 66.67% in VSCC, respectively. The overall positive rate and two two comparison had statistical significance. In the NNEDV group, the positive rate of squamous hyperplasia type and lichen sclerosus type was 35% and 25%, respectively, with no statistical significance (P>0.05), but higher than that in the normal vulva skin group (P<0.05) and lower than that in the VSCC group (P<0.05). The positive rate of HPV16-E6 in VSCC was 66. 67%. The positive rate increased with the clinical stage. The positive rate between Phase I and Phase II, and that between Phase I and Phase III had statistical significance (P<0.017), but that between Phase II and Phase III had no statistical significance (P>0.017). The positive rate gradually decreased with the tumor differentiation. The difference in well-differentiated and poorly differentiated, moderately and poorly differentiated had statistical significance (P<0.017), but that of well-differentiated and moderately differentiated had no statistical significance (P>0.017). The positive rate of lymph node metastasis VSCC was significantly higher than that of non-lymph node metastasis VSCC (P<0.05). CONCLUSION HPV infection may be an etiological factor for NNEDV. The rise of HPV16-E6 positive rate may be related to the occurrence and development of vulvar squamous cell carcinoma.
Carcinoma, Squamous Cell ; metabolism ; virology ; Female ; Humans ; Hyperplasia ; Oncogene Proteins, Viral ; metabolism ; Papillomavirus Infections ; metabolism ; Precancerous Conditions ; metabolism ; virology ; Repressor Proteins ; metabolism ; Vulvar Diseases ; metabolism ; virology ; Vulvar Neoplasms ; metabolism ; virology

OBJECTIVETo study the effect of As2O3 induction of HeLa cell apoptosis on HPV18 E6 expression and telomerase activity.

METHODSHeLa cells were treated with As2O3 in various concentrations. The effect of As2O3 on HeLa cell survival and apoptosis was determined by MTT assay, light and electron microscopy, and flow cytometry. Telomerase activity in HeLa cells was determined by TRAP-ELISA and the expression of HPV18 E6 mRNA was assayed by RT-PCR.

RESULTSAfter being treated with 2 micromol/L As2O3 for 48 h, the survival of HeLa cells decreased, and marked apoptosis was observed in a time- and dose-dependent manner. There was a good correlation between cell apoptosis and viral E6 gene expression and inhibition of telomerase activity following As2O3 treatment.

CONCLUSIONThe molecular mechanisms of As2O3 effect on HeLa cells may be related to down-regulation of HPV18 E6 oncogene expression and inhibition of telomerase activity.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; DNA-Binding Proteins ; biosynthesis ; genetics ; Down-Regulation ; HeLa Cells ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Oxides ; pharmacology ; Telomerase ; metabolism