Humans ; Squamous Cell Carcinoma of Head and Neck ; RNA, Messenger ; Immunohistochemistry ; Papillomavirus Infections/diagnosis* ; Oncogene Proteins, Viral/genetics* ; Head and Neck Neoplasms ; Cyclin-Dependent Kinase Inhibitor p16 ; Papillomaviridae ; Papillomavirus E7 Proteins/genetics* ; DNA, Viral

OBJECTIVETo investigate the expression of cyclin D1 in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma and its relationship with human papillomavirus 16 (HPV16) E7 gene expression.

METHODSBoth SiHa and Hcc94 cell lines were obtained from cervical epithelial cells of squamous cell carcinoma. E6/E7 gene was silent in Hcc94 cell line.Expression levels of cyclin D1 mRNA and protein in CIN and squamous cell carcinoma were detected by QT-PCR and immunohistochemistry (IHC) respectively. SiRNA was constructed for targeting the promoter of HPV16 E7 and then transfected into SiHa cells to establish cm-16 line with stable silencing of E7. Control cell line B3 was obtained by blank plasmid transfection into SiHa cells. RT-PCR and Western blot were used to detect cyclin D1 mRNA and protein expression in the SiHa, B3, and cm-16 cells, respectively.

RESULTSCyclin D1 was expressed in the basal cells of normal cervical squamous epithelia and the expression gradually decreased in the progression from CIN1 to CIN3. Squamous cell carcinoma showed negative or scattered expression of cyclin D1 (P<0.05). Both mRNA and protein of cyclin D1 in E7(+) SiHa cells were lower than those in cm-16 and Hcc94 cells.

CONCLUSIONSquamous cell carcinoma with high HPV E7 expression shows low level of cyclin D1, suggesting that HPV16 E7 gene inhibits the expression of cyclin D1.

Carcinoma, Squamous Cell ; metabolism ; virology ; Cell Line, Tumor ; Cervical Intraepithelial Neoplasia ; metabolism ; virology ; Cyclin D1 ; genetics ; metabolism ; Female ; Human papillomavirus 16 ; Humans ; Immunohistochemistry ; Papillomavirus E7 Proteins ; genetics ; Promoter Regions, Genetic ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Uterine Cervical Neoplasms ; metabolism ; virology

To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.
Cancer Vaccines ; biosynthesis ; genetics ; Capsid Proteins ; biosynthesis ; genetics ; Female ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; Papillomavirus Vaccines ; biosynthesis ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Uterine Cervical Neoplasms ; prevention & control ; Vaccines, DNA ; biosynthesis

OBJECTIVETo develop a prophylactic and therapeutic vaccine against human papillomavirus (HPV) type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity.

METHODSThe E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7. The recombinant HPV58 mE6E7 gene was inserted into pIRES-neo vector to generate plasmid pIRES-neo-HPV58 mE6E7. Then NIH/3T3 cell line was transfected with plasmid pIRES-neo-HPV58 mE6E7. The pIRES-neo-HPV58 mE6E7-transfected cells were the experimental group, pIRES-neo-HPV58 E6E7-transfected cells were the positive control group, and pIRES-neo empty vector-transfected cells were the negative control group. The expression of HPV58 mE6E7 protein in the experimental cells was detected by flow cytometry, immunofluorescence and Western blot. The transformation activity of HPV58 mE6E7 was tested by soft agar colony formation assay and subcutaneously tumors in nude mice. Finally, DNA vaccine was constructed with HPV58 mE6E7 fusion antigen and used to immunize C57BL/6 mice with the vaccine plasmids. The specific serum antibodies were detected by EIISA, and the number of splenic specific CD8(+) T cells secreting IFN-γ of the immunized mice was detected by ELISPOT assay.

RESULTSSequencing confirmed the expected mutation and a 100% homogeneity of the HPV58 E6E7 fusion gene. Stable transfected NIH/3T3 cells expressing HPV58 mE6E7 and HPV58 E6E7 gene were 70.3% and 84.1%, respectively. The relative expressions of HPV58 mE6E7 and HPV58 E6E7 fusion protein in 3T3-HPV58 mE6E7 experimental cells and 3T3-HPV58 E6E7 positive control cells were 2.1 ± 1.7 and 3.8 ± 1.4, respectively, and were negative in the negative control group. No colony formation was found in the experimental and 3T3-neo negative control cell groups, and 31 colonies were found in the positive control cell group, among them 10 colonies were consisted of more than 50 cells. No tumor mass was formed within 4 weeks in the nude mice of experimental and negative control groups, but among the 10 mice of positive control group tumor was formed in 6 mice. Using HPV58 mE6E7 fusion gene as target antigen of DNA vaccine, the antibody titer was 25 600, and specific immunity spots were 218.8 ± 34.4, significantly higher than that in the control group.

CONCLUSIONSThe fused and modified HPV58 E6E7 amino acid codons can abolish the transformation activity but preserve its antigenicity. HPV58 mE6E7 is a potential target gene for the development of therapeutic DNA vaccine against HPV58-associated cervical cancer.

Animals ; Cancer Vaccines ; immunology ; Capsid Proteins ; genetics ; immunology ; Cell Transformation, Neoplastic ; Cloning, Molecular ; Codon ; Female ; Immunoglobulin G ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Nude ; NIH 3T3 Cells ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomaviridae ; Papillomavirus E7 Proteins ; genetics ; immunology ; Papillomavirus Vaccines ; immunology ; Plasmids ; Point Mutation ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; Transfection ; Vaccines, DNA ; immunology

OBJECTIVETo investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.

METHODSThe HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model.

RESULTSThe codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice.

CONCLUSIONSThe data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.

Adjuvants, Immunologic ; pharmacology ; Animals ; Cancer Vaccines ; immunology ; therapeutic use ; Capsid Proteins ; genetics ; immunology ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Codon ; Escherichia coli ; immunology ; metabolism ; Female ; Humans ; Immunization ; methods ; Immunotherapy ; methods ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Oligodeoxyribonucleotides ; immunology ; Oncogene Proteins, Viral ; genetics ; immunology ; metabolism ; Papillomavirus E7 Proteins ; genetics ; immunology ; metabolism ; Papillomavirus Vaccines ; immunology ; therapeutic use ; Plasmids ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Repressor Proteins ; genetics ; immunology ; metabolism

To investigate E6 and E7 gene variations of human papillomavirus type 16 in Yunnan Province, DNA was extracted from 2000 gynecological outpatient samples. For Human papillomavirus (HPV) genotyping, the genomic DNA was first amplified by the consensus MY09/MY11 primer pair followed by nested PCR with GP5+/GP6+ primers, then the PCR products were subjected to direct DNA sequencing. A total of 20 HPV-16 viral DNAs were identified. E6 and E7 genes of HPV-16 viral DNA were then amplified using E6 and E7 specific primers, the PCR products were purified and sequenced. The results showed that mutations were found at nucleotide position 178 of HPV-16 E6 gene in 10 cases,the mutation rate was 50%; For HPV-16 E7 gene, the mutations were found at nucleotide position 647 in 10 cases; the mutation rate was 50%. Phylogenetic analysis showed that Asian (As) variants of HPV-16 were predominated in Yunnan, China. None of African-1, African-2 variants of HPV-16 was found in this region.
Adult ; Base Sequence ; China ; Female ; Genetic Variation ; Human papillomavirus 16 ; classification ; genetics ; isolation & purification ; Humans ; Middle Aged ; Molecular Sequence Data ; Mutation ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; genetics ; Papillomavirus Infections ; virology ; Phylogeny ; Repressor Proteins ; genetics

The establishment of in vitro model will provide optimal conditions for the study of human papillomavirus (HPV)-associated cervical cancer. In this study, E6 and E7 gens of HPV31 were cloned and expressed in E. coli. The recombinant proteins were purified and used as antigens to immunize mice for the production of polyclonal antibody. Mammalian expression plasmid pBudCE4. 1-HPV31-E6/E7 was also constructed and transfected into C33A cells. The transfected cells were then selected by Zeocin. The expressions of the E6 and E7 mRNAs and proteins were detected by RT-PCR and Western blot respectively. A stable cervical cancer cell line was established as an in vitro model for the study of human papillomavirus type 31(HPV31) associated cervical cancer.
Animals ; Cell Line ; virology ; Female ; Human papillomavirus 31 ; genetics ; metabolism ; Humans ; Mice ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus E7 Proteins ; genetics ; metabolism ; Papillomavirus Infections ; virology ; Recombinant Proteins ; genetics ; metabolism ; Transfection

HPV16 E7 fusion protein was expressed in E. coli BL21, and its applied value for HPV was evaluated. HPV16 E7 gene was amplified by PCR, and cloned into prokaryotic expression vector pGEX6p-1. The recombinant plasmid was transformed into E. coli BL21, and HPV16 E7 fusion was expressed through IPTG induction. The expressed product was analyzed by SDS-PAGE and Western blot, subsequently purified according to Glutathione Sepharose 4B purification procedure. An indirect ELISA with the purified fusion protein as the coating antigen was then established to detect E7 serum antibodies from mice immunized with recombinant Listeria monocytogenes delivering HPV16 E7. The results demonstrated that the soluble fusion protein was highly expressed at 25 degrees C after induction with 0.5 mM IPTG. Furthermore, the result of Western blot analysis showed that the fusion protein had good specific reaction with an anti-E7 monoclonal antibody. Indirect ELISA result confirmed that the fusion protein could detect the serum antibodies against E7 with a titer of 1:200. The expressed GST-E7 fusion protein was immunocompetent, which was useful in the research of E7 biological function and therapeutic vaccine.
Animals ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Female ; Mice ; Mice, Inbred C57BL ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; isolation & purification ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; isolation & purification

To study the gene variation and the distribution of HPV16 variant in Hubei, China, DNA was extracted from cervical cancer tissue samples. The E6 and E7 genes of HPV16 were amplified and the PCR products were sequenced using E6- and E7-specific primers. Fortyseven cases were found mutations at nucleotide position 178 of HPV16 E6 gene in 80 cervical cancer samples. This mutation resulted in amino acid change from Asp to Glu. The rate of mutation at nucleotide position 178 of E6 gene was 58. 75%. Twenty two cases were found mutations at nucleotide position 647 of HPV16 E7 gene in 31 cervical cancer samples. This mutation resulted in amino acid change from Asn to Ser. The rate of mutation was 70.97%. These results showed that mutations at nucleotide position 178 of E6 gene, nucleotide position 647 of E7 gene of HPV16 in cerveical cancer samples were prevalent in Hubei, China. Phylogenetic analysis showed that Asian (As) variants of HPV16 are predominated in Hubei, China. European (Ep) varinats were also found in Samples in Hubei areas. None of Asian American (AA), African-1 (Af-1), African-2 (Af-2) variants of HPV16 was found in this region. Whether Asian (As) variants of HPV16 are more oncogenic and play a much more important role in the progress of cervical cancer than European (Ep) variants is not clear. More sequences of E6 and E7 gene in CIN and normal cervical tissue samples and study of the function of E6 and E7 protein of these HPV16 variants are needed to adress above question.
Adult ; China ; Evolution, Molecular ; Female ; Humans ; Middle Aged ; Mutation ; Oncogene Proteins, Viral ; chemistry ; classification ; Papillomavirus E7 Proteins ; classification ; genetics ; Phylogeny ; Polymerase Chain Reaction ; Repressor Proteins ; chemistry ; classification ; Uterine Cervical Neoplasms ; virology

OBJECTIVEThe aim of this study was to analyze the relative distribution and gene variation of HPV16 transforming gene E6, E7 and E5 at different stages of cervical lesions.

METHODSDNA was extracted from tissue samples of 200 patients with cervical lesions, including 124 cervical cancer, 17 CIN grade I and II, 23 CIN grade III and 36 cervicitis. Then HPV16 E6, E7 and E5 genes were amplified, and part of the E6 and E7 PCR products were sequenced using the HPV16 E6 and E7 specific primers.

RESULTSThe positive rate of E6 gene in cervicitis, CINI and CINII, CINIII and cervical cancer was 25.0%, 29.4%, 60.9% and 76.6%, respectively. The positive rate of E7 gene was 16.7%, 41.2%, 43.5% and 61.3%, respectively. The positive rate of E5 gene was 5.6%, 5.9%, 30.4% and 40.3%, respectively. HPV16 E6 gene mutations in Nt 178 were found in 47 case from 80 cervical cancer samples, resulting in amino acid change of Asp to Glu. The mutation rate was 58.8%.Otherwise the mutation rate of E6 178 in cervicitis and CIN I approximately III samples was 25.0% and 31.8%. E7 mutations were found in Nt 647 in 21 cervical samples from 30 cervical cancer samples, resulting in amino acid change of Asn to Ser. The mutation rate was 70.0%. The mutation rate of E6 647 in cervicitis and CINI approximately III samples was 35.0% and 40.9%, respectively.

CONCLUSIONThe positive rate of E6 and E7 increase gradually from cervicitis, CINI and CINII, CINIII to cervical cancer. The rate of E5 is relatively lower than that of E6 and E7 gene in cervical tissue samples. These results show that E6 and E7 gene are highly associated with the progress of cervical cancer and E5 genes are lost in the development of cervical cancer. High frequency mutations of HPV16 E6 and E7 gene in E6 178, E7 647 have been found in cervical cancer samples in Hubei province, China. These results approved that the HPV16 variants prevalent in this area are different from the European and African variants.

Adult ; Carcinoma ; metabolism ; virology ; Cervical Intraepithelial Neoplasia ; metabolism ; virology ; China ; Female ; Human papillomavirus 16 ; genetics ; Humans ; Middle Aged ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus E7 Proteins ; genetics ; metabolism ; Papillomavirus Infections ; genetics ; Point Mutation ; Repressor Proteins ; genetics ; metabolism ; Uterine Cervical Neoplasms ; metabolism ; virology ; Uterine Cervicitis ; metabolism ; virology