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El objetivo de este estudio fue determinar la presencia del Virus Papiloma Humano (VPH) tipo 16 y 18 en biopsias de tejido mamario parafinado de pacientes con diagnóstico clínico de cáncer de mama. Se analizaron 32 biopsias de cáncer de mama embebidas en parafina para detectar el ADN de VPH mediante PCR en tiempo real, los iniciadores estuvieron dirigidos al gen E6. Se evaluaron el tipo histológico, grado histológico y la sobreexpresión de C-erB2 y Ki-67 mediante inmunohistoquímica. El 84,38% (27) fueron positivos para VPH, el 25% (8) fueron positivos para VPH-16 y el 59,38% (19) para VPH-18. El 15,63% (5) de las muestras presentaron infección mixta. Se evidenció la sobrexpresión de C-erbB2 y Ki-67 en 6,25% (2) de las muestras positivas para VPH-16 y 15,63% (5) de las muestras positivas para VPH-18. Se detectó ADN de VPH-16 y VPH-18 en las muestras de biopsias analizadas mediante PCR en tiempo real.
The aim of this study was to determine the presence of Human Papillomavirus (HPV) type 16 and 18 in biopsies of paraffin-embedded breast tissue from patients with clinically diagnosed breast cancer. 32 paraffin-embedded breast cancer biopsies were analyzed in order to detect HPV DNA by real-time PCR, the primers were directed at the E6 gene. The histological type, histological grade and overexpression of C-erB2 and Ki-67 were evaluated by immunohistochemistry. 84.38% (27) of the samples were positive for HPV, 25% (8) were positive for HPV-16 and 59.38% (19) were positive for HPV-18. Mixed infection was found in 15.63% (5) of the samples. Overexpression of C-erbB2 and Ki-67 was seen in 6.25% (2) of the samples positive for HPV-16 and in 15.63% (5) samples positive for HPV-18. HPV-16 and HPV-18 DNA was detected in the biopsy samples analyzed by real-time PCR.
Subject(s)
Humans , Female , Breast Neoplasms , Human papillomavirus 16 , Human papillomavirus 18 , Papillomaviridae , Tissues , Biopsy , Immunohistochemistry , Clinical Diagnosis , Polymerase Chain ReactionABSTRACT
Objectives: Squamous cell carcinoma (SCC) of the cervix is one of the leading causes of death in developing countries. Infection with high-risk human papillomavirus (HR-HPV) is the major risk factor to develop malignant lesions HR types (HPV16 and HPV18) account for about 70% of all invasive cervical cancers worldwide. It is estimated that 833 Sudanese women are diagnosed with cervical cancer and 534 die from the disease every year. The present study aimed to detect HPV 16, and determine the association of HPV16 with age and various grades of cervical carcinoma in patients with clinically confirmed cervical SCC. Materials and Methods: A total of 158 formalin fixed paraffin embedded tissues blocks from Sudanese women diagnosed as cervical cancer and benign were collected between 2012 and 2016 at Omdurman Maternity Hospital and National Laboratories, Khartoum, Sudan. HPV DNA detection was done using HPV 16 specific primers in real-time polymerase chain reaction. Results: The frequency of HPV 16 was identified among 10.34% (n = 6) and 6% (n = 6) women with abnormal cytology and normal cytology, respectively. Based on age, high prevalence rate of HPV 16 was observed among age group 61–70 in women with malignant cases. The degree of differentiation, an important classification in SCC cases revealed that 5% (n = 3) cases had moderately differentiated SCC and two of them were keratinized SCC. In addition, 3.4% (n = 2) SCC cases were keratinized and well differentiated. Conclusion: Overall, the prevalence of HPV types 16 was higher but had no significant association with cervical SCC in Sudanese women
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Objective To investigate the effect of heterogeneous nuclear ribonucleoprotein K (hnRNP K) and its interaction with human papillomavims 16 (HPV16) on cervical intraepithelial neoplasia (CIN).Methods The participants included 67 women with normal cervix (NC),69 women with CIN Ⅰ and 68 women with CIN Ⅱ/Ⅲ in a community cohort of pathologically diagnosed women established in Jiexiu of Shanxi province,from June 2014 to June 2015.A structured questionnaire was used to collect the demographic data of the subjects and the related factors of cervical lesions.Cervical exfoliated cells and cervical tissues from biopsy or surgery were selected.The infection status of HPV16 was detected by flow-through hybridization.The protein expression levels of hnRNP K were evaluated by Western blot.SPSS 23.0 software was used to collate and analyze the data.To study the differences in demographic characteristics,related factors,hnRNP K protein and HPV16 infection among NC,CIN Ⅰ and CIN Ⅱ / Ⅲ groups,X2 test,trend x2 test,and Kruskal-Wallis H test were conducted.Multiple comparisons of hnRNP K protein in three groups were completed by using the Bonferroni method.The OR and its 95％CI of hnRNP K,HPV16 and CIN were calculated by using the unconditional logistic regression models.Two-way interactions between hnRNP K protein and HPV16 infection on CIN were analyzed by using additive model and related indicators.Results HPV16 infection rates were 10.4％ in women with normal cervix,14.5％ in women with CIN Ⅰ and 41.2％ in women with CIN Ⅱ/Ⅲ,respectively.The differences among three groups were significant (P＜0.001).Moreover,the infection rates of HPV16 gradually increased with the increasing severity of CIN (trend x2=18.512,P＜0.001).The differences in protein expression of hnRNP K among three groups were significant (H=48.138,P＜0.001) and the expressionincreased with the development of cervical lesionss (trend x2=21.765,P＜0.001).Results from the interaction analysis indicated that there were additive effects between high expression of hnRNP K protein and HPV16 in CIN Ⅱ/Ⅲ group compared with normal group (API=0.639,95％CI:0.083-1.196).In contrast,no such additive effect was found in CIN Ⅰ group.Conclusions HPV16 infection and over-expression of hnRNP K protein were associated with the increased risk of cervical intraepithelial neoplasia.There might be interaction between hnRNP K protein overexpression and HPV16 infection existed on the progress of CIN Ⅰ/Ⅲ.
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Objective To investigate the effect of heterogeneous nuclear ribonucleoprotein K (hnRNP K) and its interaction with human papillomavims 16 (HPV16) on cervical intraepithelial neoplasia (CIN).Methods The participants included 67 women with normal cervix (NC),69 women with CIN Ⅰ and 68 women with CIN Ⅱ/Ⅲ in a community cohort of pathologically diagnosed women established in Jiexiu of Shanxi province,from June 2014 to June 2015.A structured questionnaire was used to collect the demographic data of the subjects and the related factors of cervical lesions.Cervical exfoliated cells and cervical tissues from biopsy or surgery were selected.The infection status of HPV16 was detected by flow-through hybridization.The protein expression levels of hnRNP K were evaluated by Western blot.SPSS 23.0 software was used to collate and analyze the data.To study the differences in demographic characteristics,related factors,hnRNP K protein and HPV16 infection among NC,CIN Ⅰ and CIN Ⅱ / Ⅲ groups,X2 test,trend x2 test,and Kruskal-Wallis H test were conducted.Multiple comparisons of hnRNP K protein in three groups were completed by using the Bonferroni method.The OR and its 95％CI of hnRNP K,HPV16 and CIN were calculated by using the unconditional logistic regression models.Two-way interactions between hnRNP K protein and HPV16 infection on CIN were analyzed by using additive model and related indicators.Results HPV16 infection rates were 10.4％ in women with normal cervix,14.5％ in women with CIN Ⅰ and 41.2％ in women with CIN Ⅱ/Ⅲ,respectively.The differences among three groups were significant (P＜0.001).Moreover,the infection rates of HPV16 gradually increased with the increasing severity of CIN (trend x2=18.512,P＜0.001).The differences in protein expression of hnRNP K among three groups were significant (H=48.138,P＜0.001) and the expressionincreased with the development of cervical lesionss (trend x2=21.765,P＜0.001).Results from the interaction analysis indicated that there were additive effects between high expression of hnRNP K protein and HPV16 in CIN Ⅱ/Ⅲ group compared with normal group (API=0.639,95％CI:0.083-1.196).In contrast,no such additive effect was found in CIN Ⅰ group.Conclusions HPV16 infection and over-expression of hnRNP K protein were associated with the increased risk of cervical intraepithelial neoplasia.There might be interaction between hnRNP K protein overexpression and HPV16 infection existed on the progress of CIN Ⅰ/Ⅲ.
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OBJECTIVE: Human papillomavirus (HPV) 16 is the most carcinogenic HPV genotype. We investigated if HPV16 L1 capsid protein and E2/E6 ratio, evaluated by cervical cytology, may be used as biomarkers of ≥cervical intraepithelial neoplasia (CIN) 2 lesions. METHODS: Cervical specimens were obtained from 226 patients with HPV16 single infection. Using cytology specimen, L1 capsid protein and E2/E6 ratio were detected and the results were compared with those of the conventional histologic analysis of cervical tissues (CIN1–3 and squamous cell carcinoma [SCC]) to evaluate the association. RESULTS: The L1 positivity of CIN2/3 was significantly lower than that of normal cervical tissue (p < 0.001) and SCC demonstrated significantly lower L1 positivity than CIN1 (p < 0.001). The mean E2/E6 ratios of specimens graded as SCC (0.356) and CIN2/3 (0.483) were significantly lower than those of specimens graded as CIN1 (0.786) and normal (0.793) (p < 0.05). We observed that area under the receiver operating characteristic curve (AUC) for E2/E6 ratio (0.844; 95% confidence interval [CI]=0.793–0.895) was higher than that for L1 immunochemistry (0.636; 95% CI=0.562–0.711). A combination of E2/E6 ratio and L1 immunocytochemistry analyses showed the highest AUC (0.871; 95% CI=0.826–0.917) for the prediction of ≥CIN2 lesions. CONCLUSION: To our knowledge, this is the first study to validate HPV L1 capsid protein expression and decreased HPV E2/E6 ratio as valuable predictive markers of ≥CIN2 cervical lesions. Cervical cytology may be analyzed longitudinally on an outpatient basis with noninvasive procedures as against invasive conventional histologic analysis.
Subject(s)
Humans , Area Under Curve , Biomarkers , Capsid Proteins , Carcinoma, Squamous Cell , Uterine Cervical Dysplasia , Epithelial Cells , Genotype , Immunochemistry , Immunohistochemistry , Outpatients , ROC Curve , Squamous Intraepithelial Lesions of the Cervix , Uterine Cervical Neoplasms , Virus IntegrationABSTRACT
Background and purpose:The incidence of cervical cancer is rather high in Xinjiang, which is closely associated with the infection of human papilloma virus type 16 (HPV-16). The purpose of this study was to analyze the variants and function of HPV-16 upstream regulatory region (URR) in the tissues of cervical cancer biopsies from Xinjiang.Methods:The DNAs were extracted from the tissues of cervical epithelial atypical hyperplasia (CIN) and cervical cancer biopsies. HPV-16URR segments were ampliifed by PCR. Based on the sequence analysis of the URR, the representativeURR variants were selected and cloned into pGL3-Basic. The recombinant plasmids were transfected into Vero cell lines respetively. Luciferase activity of transfected cells was detected 48 h after transfection. Results:Fifty-ifve HPV-16URR DNA fragments were obtained through PCR, and 44 mutations were found from the URR fragments. 4 of these mutations, including nt7192(G→T) , nt7433(- →T), nt7435 (C→G) and nt7863 (A→-) occurred in all sequences. The mutation at nt7520 (G→A) occurred in 54URR sequences, and the 39 other mutations were present in different samples. Based on the location and frequency of the mutations in theURR fragments, 9URR variants were selected and cloned into pGL3-Basic. Then the luciferase activity of the cells transfected with pGL3-URR plasmids was detected respectively. Promoter activity ofURR mutants from cervical cancer are significantly higher than that ofURR mutants from CIN (P<0.01). Promoter activity ofURR fragments from some cervical cancer was signiifcantly higher than that of theURR fragments from SiHa and Caski cells.Conclusion:Multiple mutations occurred in HPV-16URR of cervical cancer patients from Xinjiang. The promoter activity and carcinogenicity of some URR mutants have been enhanced.
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Objectives: To analyze the immunogenicity of virus-like particles (VLP) of human papillomavirus type 16 (HPV16) isolated in East China and the adjuvant potential of interleukin-12 (IL-12). Methods: The variant HPV16 L1VLP expressed in sf9 insect cells were purified with cesium chloride gradient centrifugation. BALB/c mice were vaccinated with VLP (L1N), VLP with Freund's adjuvant (L1A) or VLP with IL-12 recombinant plasmid (L1P). HPV16 VLP specific IgG and IFN-γ level in the serum were detected by ELISA, and the percentage of CD4+ and CD8+ in spleen cells was detected with flow cytometry. Results: The titers of serum IgG antibodies in vaccinated groups were higher than in negative control and the serum antibodies mainly recognized conformation-dependent HPV16 VLP epitopes. Splenic CD4+ and CD8+ T cell subsets increased after vaccination in every experimental group, and CD8+ increased obviously in L1P group. The ratio of CD4+/CD8+ decreased in L1P group and increased in the other two groups, compared to control group. Vaccination induced specific secretion of IFN-γ in the serum of vaccinated group (p < 0.05), especially in the L1P group. Conclusions: VLP of HPV16 variant strain isolated in East China could induce humoral immunity and cellular immunity in mice, and IL-12 recombinant plasmid can enhance cellular immunity. .
Subject(s)
Animals , Female , Humans , Mice , /immunology , /blood , /genetics , Papillomavirus Infections/prevention & control , Vaccines, Virus-Like Particle/immunology , Adjuvants, Immunologic , Antibodies, Viral/blood , Antibodies, Viral/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Immunity, Cellular/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , /immunology , Mice, Inbred BALB C , Molecular Sequence Data , Papillomavirus Infections/immunologyABSTRACT
Objective To clone human papillomavirus type 16 genome from Anhui province, analyze genome se-quence of HPV16 and study its genomic characteristics. Methods Five pathological specimens of cervical cancer from Anhui province were collected and the total DNA was extracted. Specific primers were designed to clone HPV16 genome in four fragments. The sequence of four fragments was assembled manually and nucleotide sequence was analyzed after sequencing. Results A cervical pathological sample containing HPV16 was detected and the genome sequence with full length of 7 906 nts (GenBank accession number:KC935953) was obtained. Sequence alignment of genome nucleotide sequence showed that HPV16 of Thailand and HPV16 of Japan were more similar to HPV16 of Anhui (HPV16-Anhui) than other HPV genome nucleotide sequence, their similarity reached 99. 5%. Phylogeny tree analysis demonstrated that HPV16-Anhui and other 7 HPV16 clustered into a single branch. Con-clusion HPV16 genome nucleotide sequence is obtained from Anhui province for first time with great significance for further understanding of HPV16 variation from Anhui even east China.
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Aims: Human papillomavirus type 16 (HPV16) is the primary etiological agent of cervical cancer. The variations in the amino acid sequence of the HPV16 E6 and E7 oncoproteins are known to correlate with both their oncogenic potential and geographic distribution. Study Design: The present study was designed to analyze sequence variations in E6 and E7 genes of HPV16 in order to evaluate the intratype variants circulating in our population. Methodology: The entire E6 and E7 genes of 31 HPV16 isolates from Moroccan patients with cervical cancer were sequenced and analyzed. Results: Sequence analysis of HPV16-E6 showed a high prevalence (64.5%) of the African lineage. The European and the North-American variants were detected in respectively 19.4% and 16% of the HPV16 positive specimens. At the amino acid level, the most prevalent missense mutations revealed in the E6 gene were H78Y, Q14D, L83V, R10I and Q14H. Our data also showed that E7 appeared to be better conserved as compared to E6, with a high frequency of two silent variations at G789A and T795C nucleotides and one hot spot of E7 nucleotide variation A647G leading to N29S. Conclusion: The present study provides a new data on the genetic diversity of HPV16 and highlights the possible association between the high prevalence of HPV16 African variants and the high incidence of cervical cancer in Morocco.
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ObjectiveTo construct one recombinant adenovirus AdE7E6 expressing HPV16 E6 and E7 fusion protein as candidate for HPV16 therapeutic vaccine.MethodsThe codon-optimized E6 and E7 gene,were fused to create one open reading frame,then inserted into adenovirus vector pCD316.A strain of recombinant adenovirus was constructed through homologous recombinant in 293 cells,and identified by PCR and Western blot.Finally,it was employed to study it's immunogenicity and the activity of the tumor growth regression.ResultsThe PCR result showed that E6E7 fusion gene had been integrated in recombinant Ad5 DNA.Western blot test confirmed that the E6E7 fusion protein was highly expressed in 293 cells infected with Ad5E7E6 recombinant adenovirus.The recombinant adenovirus elicited significant E7 specific CD8+ T lymphocyte response in vaccinated mice.These responses could completely prevent the TG-1 tumor cell bearing mice treated with AdE7E6 from developing into tumor.ConclusionThese results suggested that rAd5E7E6 could be a potent vaccine candidate for the treatment of HPV16-associated tumors and their precancerous transformations.
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Objective To select an optimal non-specific antigen blocking method by using immuno-infiltration assay so as to suit protein chip preparation. Methods Human papillomavirus type 16 L1 protein expressed by insect-baculovirus espressin system was incubated with skimmed milk powder, calf serum, bovine serum albumin (BSA) combinations of five kinds of methods to block the non-specific antigen. PBS was used as control. The effect of eliminating non-specific stain was detected by immuno-infiltration assay. Results After repeated tests, the results showed that the stability and repeatability of blocking effects were poor for the fixing up antigen first and then blocking method, and the blank control was prone to false positive. The infiltration rate of NC membrane would be affected by using skimmed milk powder as a blocking agent because the pore of NC membrane was easily plugged by milk powder particles. The use of calf serum as a blocking agent made it very difficult to determine the result because the calf serum absorbed by NC membrane produced the background; however, when 20g/L BSA was used to blocking before fixing up antibody, the results became satisfactory. Conclusion Fixing up antibody after blocking in immuno-infiltration assay showed that the blocking effect against non-specific antigen was satisfactory, stable and repeatable, indicating this method is a novel optimal blocking method compared with others.
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Background & objective: Recombinant DNA technology allows expression of the human papillomavirus (HPV) major capsid protein (L1) in heterologous expression systems and the recombinant protein self assembles to virus-like particles (VLP). We took up this study to produce recombinant HPV-16 L1 in yeast, establish the process of recombinant L1 derived VLP preparation and develop an ELISA using VLP as the antigen for serological evaluation of anti HPV-16 L1 antibody status. Methods: Complete HPV-16 L1 was amplified from genomic DNA of an esophageal cancer biopsy, cloned and the protein was expressed in a galactose-inducible Saccharomyces cerevisiae expression system. Self assembled VLP was purified by a two-step density gradient centrifugation process and the VLP preparation used to test its suitability in developing an ELISA. Results: The recombinant protein was predominantly a ~55 KD species with distinct immunoreactivity and formed VLP as confirmed by electron microscopy. An ELISA using the VLP showed its efficacy in appropriate immunoreactivity to serum/plasma IgG. Interpretation & conclusions: Recombinant HPV-16 capsid protein derived VLP was produced and the VLP antigen based ELISA can be used to probe serological association of HPV with different clinical conditions. The VLP technology can be improved further and harnessed for future vaccine development efforts in the country.
Subject(s)
Antibodies, Viral/analysis , Antigens, Viral/biosynthesis , Antigens, Viral/genetics , Capsid Proteins/biosynthesis , Capsid Proteins/genetics , Capsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Human papillomavirus 16/genetics , Human papillomavirus 16/immunology , Human papillomavirus 16/ultrastructure , Humans , Oncogene Proteins, Viral/biosynthesis , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Saccharomyces cerevisiae/geneticsABSTRACT
More than 99% of cervical cancers have been associated with human papillomaviruses (HPVs), particularly HPV type 16. The clear association between HPV infection and cervical cancer indicates that HPV serves as an ideal target for development of preventive and therapeutic vaccines. Although the recently licensed preventive HPV vaccine, Gardasil, has been shown to be safe and capable of generating significant protection against specific HPV types, it does not have therapeutic effect against established HPV infections and HPV-associated lesions. Two HPV oncogenic proteins, E6 and E7, are consistently co-expressed in HPV-expressing cervical cancers and are important in the induction and maintenance of cellular transformation. Therefore, immunotherapy targeting E6 and/or E7 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. It has been established that T cell-mediated immunity is one of the most crucial components to defend against HPV infections and HPV-associated lesions. Therefore, effective therapeutic HPV vaccines should generate strong E6/E7-specific T cell-mediated immune responses. DNA vaccines have emerged as an attractive approach for antigen-specific T cell-mediated immunotherapy to combat cancers. Intradermal administration of DNA vaccines via a gene gun represents an efficient way to deliver DNA vaccines into professional antigen-presenting cells in vivo. Professional antigen-presenting cells, such as dendritic cells, are the most effective cells for priming antigen-specific T cells. Using the gene gun delivery system, we tested several DNA vaccines that employ intracellular targeting strategies for enhancing MHC class I and class II presentation of encoded model antigen HPV-16 E7. Furthermore, we have developed a strategy to prolong the life of DCs to enhance DNA vaccine potency. More recently, we have developed a strategy to generate antigen-specific CD4+ T cell immune responses to further enhance DNA vaccine potency. The impressive pre- clinical data generated from our studies have led to several HPV DNA vaccine clinical trials.
Subject(s)
Female , Humans , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Papillomavirus Infections/immunology , Papillomavirus Vaccines/administration & dosage , Repressor Proteins , Uterine Cervical Neoplasms/prevention & control , Vaccines, DNA/administration & dosage , Viral Vaccines/administration & dosageABSTRACT
Template direct dye-terminator incorporation with fluorescence-polarization (TDI-FP assay) is a technology for genotyping single nucleotide polymorphisms (SNPs). To apply this method in analyses of A647G variation in human papillomavirus (HPV) 16 E7 gene from HPV 16-positive cervical tissues, a total of 91 and 49 HPV 16-positive DNA samples obtained from women with cervical cancer and normal/inflamed cervices living in Shaanxi in northwest China were subjected to the partial E7 gene PCR with nucleotide (nt) 647 in the products. Then, the oligonucleotide probe designed to anneal immediately to nt 647 was hybridized to the template within the PCR amplicons, and extended specifically by TAMRA-ddTTP or R110-ddCTP directed by the base at nt 647. The increasing FP values were read and the base at nt 647 was identified. The prevalence of nt 647 A→G was 35.71% (50/140). The variation 647G detected in 42.86% (39/91) of women with cervical cancer was significantly higher than 22.45% (11/49) detected in those with normal/inflamed cervices (x2 = 5.778, P = 0.016). The odds ratio (OR) between these two groups was 2.59 (95% confidence interval=l.17～5.71). The results demonstrate that TDI-FP method can be potentially applied in analysis of interest point mutations in HPVs. The incidence and risk implication of HPV 16 A647G variant infection in Shaanxi, China, displays significant geographic difference from other areas. The HPV 16 with E7 gene A647G point mutation appears to have a higher risk for invasive cervical cancer in women living in Shaanxi.
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Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.
Subject(s)
Humans , Asparagine , Baculoviridae , Blotting, Western , Chromatin , Cytoplasm , Immunoprecipitation , Insecta , Open Reading Frames , Phosphoproteins , Serine , Sf9 CellsABSTRACT
Recent studies of molecular biology have suggested that infection with human papillomavirus(HPV) is implicated in the pathogenesis of cervical carinoma. HPV infection alone, however, does not appear to be sufficient for the process of maliganant transformation, suggesting the requirement of additional cellular events. The mutation of p53, which is involved in negative control of cell proliferation, may play a role in the carcinogenesis of cervical carcinoma. The present study was designed to clarify the association between infection with HPV and p53 alteration in primary carcinoma of human uterine cervix. We investigated 46 prim-ary cervical carcinomas for the presence of HPV DNA by in situ hybridization(ISH) with probe specific for HPV 16/18, and examined the accumulation of p53 protein by immunohis-tochemistry(IHC) and the p53 alteration by polymerase chain reaction-single strand confor-mation polymorphism(PCR-SSCP) using formalin fixed, paraffin -embedded tissue. HPV DNA 16/18 was detected in 18 cases(39.1%) of 46 cervical carcinomas. The accumulation of p53 was identified in tumor cells: low level 43.5%(20/46) and high level 32.6% difference of positive reaction by IHC method. But there was no statistical significant between the infection of HPV and the accumulation of p53(p=0.847). Mutations in exons 4 through 9, where the vast majority of point mutations were reported in human neoplasms, were screened by PCR-SSCP analysis. Altered mobilities of the PCR product of p53 were also found in 9 cases(26.5%) of 34 cervical carcinoma: one in exon 4, four in exon 5/6, two in exon 7, and two in exon 8/9. The mutation of p53 was observed in 41.1%(19/46) respective of the result of IHC and PCR-SSCP, and there was slightly higher p53 alteration in HPV negative cases(23.8%, 11/46) than in HPV positive cases(17.4%, 8/46) without statistical significance(p=0.729). The conclusion of these observations suggests that HPV infection and alteration of p53 may play a critical role in tumorigenesis of carcinoma of the human uterine cervix independently, ant there is important difference in the tumorigenic pathway between two factors.
Subject(s)
Female , Humans , Ants , Carcinogenesis , Carcinoma, Squamous Cell , Cell Proliferation , Cervix Uteri , DNA , Exons , Formaldehyde , Genes, p53 , Molecular Biology , Paraffin , Point Mutation , Polymerase Chain ReactionABSTRACT
Over 60 different types of human papillomavirus (HPV) have been identified, and they are classified into high and low risk groups based on the risk for malignant progression of HPV associated lesions. HPVs belonging to a high risk group have been shown to express two major transforming proteins, E6 and E7. With respect to the transforming activity of these proteins, many investigators have reported the location of these proteins in the cell, but their results are still controversial. In the present study, HPV type 16 E6 or E7 open reading frame (ORF) proteins were expressed and localized in human epidermal keratinocytes (RHEK-1) using the vaccinia virus as an expression vector. Immunofluorescence detection using monoclonal antibodies against E6 or E7 ORF proteins revealed that E6 or E7 proteins of HPV type 16 were located in the cytoplasm of RHEK-1 cells. These results suggest that E6 and E7 proteins bind to the tumor suppressor counterparts, thereby preventing transport of these proteins into the nucleus. These antioncogene products that fail to be rapidly transported out of the cytosol may be degraded by certain proteases such as the ubiquitin dependent system. In this way, the precise function of antioncogene products in the regulation of cell growth could be destroyed, and abnormal cell growth could occur.