Presença do papilomavirus humano tipo 16 e expressão gênica da proteína p16ink4a e oncoproteína e7 no carcinoma colorretal / Hpv16 and expression of protein p16ink4a and e7 oncoprotein in colorectal carcinoma

RESUMO - INTRODUÇÃO: O papilomavírus humano (HPV) é agente das doenças sexualmente transmissíveis de maior prevalência no mundo que estão associadas ao câncer do colo do útero e canal anal. A ação do HPV na carcinogênese colorretal não está ainda estabelecida. OBJETIVO: Estudar a eventual correlação entre a presença do HPV tipo 16 e a expressão gênica da proteína p16INK4a e da oncoproteína E7 de HPV e de seus níveis no tecido do carcinoma colorretal. METODOS: Estudo retrospectivo caso-controle de 79 doentes com carcinoma colorretal divididos em dois grupos: HPV presente e HPV ausente. Foi realizada reação em cadeia da polimerase (PCR), além da hibridização do tipo dot blot para o HPV 16 e o HPV 18 Amostras do tecido colorretal também foram submetidas ao estudo imuno-histoquimico para avaliar o nível tecidual das proteínas E7 e p16INK4a. RESULTADOS: O HPV foi identificado em 36 (45,6%) casos. Não houve diferença significante entre os grupos quanto ao sexo (p=0,056), idade (p=0,1), localização cólica e/ou retal (0,098) e presença do HPV. A expressão gênica da oncoproteína E7 de HPV estava presente em 3,12% dos casos (p=0,9) e a expressão da proteína p16INK4a foi observada em 46,3% (p=0,27) dos indivíduos com detecção do HPV. CONCLUSÃO: A expressão gênica e os níveis teciduais da oncoproteína E7 e da proteína p16INK4a encontrados nos pacientes positivos para o HPV sugerem a ausência de atividade oncogênica do HPV tipo 16 no carcinoma colorretal.

ABSTRACT - BACKGROUND: Human papillomavirus (HPV) is the agent of the most prevalent sexually transmitted diseases in the world associated with cervix and anal canal cancer. The action of HPV on colorectal carcinogenesis is not yet established. OBJECTIVE: This research aimed to study the possible correlation between the presence of HPV16 and the gene expression of p16INK4a protein and HPV E7 oncoprotein and their levels in colorectal carcinoma tissue. METHODS: A retrospective case-control study of 79 patients with colorectal carcinoma was divided into two groups: HPV-positive and HPV-negative. The polymerase chain reaction was performed, in addition to dot-blot hybridization for HPV16 and HPV18. Colorectal tissue samples were also subjected to immunohistochemical study to assess the tissue level of E7 and p16INK4a proteins. RESULTS: HPV was identified in 36 (45.6%) cases. There was no significant difference between groups regarding gender (p=0.056), age (p=0.1), colic and/or rectal location (0.098), and presence of HPV. Gene expression of HPV E7 oncoprotein was present in 3.12% of cases (p=0.9), and p16INK4a protein expression was observed in 46.3% (p=0.27) of those selected with HPV detection. CONCLUSION: Gene expression and tissue levels of E7 oncoprotein and p16INK4a protein found in HPV-positive patients suggest the absence of HPV16 oncogenic activity in colorectal carcinoma.

Association between the p53 arginine/arginine homozygous genotype at codon 72 and human papillomavirus E6/E7 mRNA expression

ABSTRACT Objective: To evaluate the association between p53 polymorphisms and human papillomavirus (HPV) E6/E7 mRNA expression. Methods: We analyzed 175 cervical samples from women aged 16-69 years old who were tested for HPV E6/E7 mRNA expression (NucliSENS® EasyQ® HPV). The samples were divided into three groups: positive (n = 75) those with positive HPV E6/E7 mRNA expression and positive high-risk HPV Hybrid Capture (HR-HC) test; negative (n = 52) those with negative HPV E6/E7 mRNA expression and positive HR-HC; and control (n = 48) those with negative HPV E6/E7 mRNA expression and negative HR-HC. The p53 polymorphisms at codons 11, 72, and 248 were evaluated through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: The frequency of the arginine/arginine homozygous genotype at codon 72 was significantly higher in the positive (49.3%) than in the negative (32.7%) and control groups (20.8%, p = 0.002*). The frequency of the arginine allele was also significantly higher in the positive (67.3%) than in the negative (53.8%) and control groups (38.5%, p < 0.001*). The arginine/arginine homozygous genotype was significantly associated with positive HPV E6/E7 mRNA expression (positive group) compared with negative and control groups (odds ratio: 2.633; 95% CI, 1.399-4.954, p = 0.003). The frequency of arginine/arginine homozygous genotype at codon 72 remained significantly more frequent in the positive group of women aged ≥30 years than in the other two groups. Conclusion: The presence of the p53 arginine/arginine homozygous genotype at codon 72 was significantly associated with the positive HPV E6/E7 mRNA expression.

Antibody detection against HPV16 E7 & GP96 fragments as biomarkers in cervical cancer patients.

Background & objectives: Cervical cancer is the second most frequent cancer among females worldwide, especially human papilloma viruses (HPV) types 16 and 18. In viral systems the identification of serological markers would facilitate the diagnosis of HPV infections and virus-related disease. The aim of the present investigation was to determine and search for serologic markers in cervical cancer patients associated with HPV. Methods: A total of 58 Iranian women with invasive cervical carcinoma including adenocarcinoma and squamous cell carcinoma (SCC) were included. Serum antibody response to HPV infections in patients was detected by Western blot and ELISA techniques based on recombinant HPV16E7 and the N-terminal and C-terminal fragments of gp96 (NT-gp96 and CT-gp96) proteins. These recombinant proteins were expressed in Escherichia coli as a His-tag protein and purified using affinity chromatography. Results: The ELISA results indicated that patients with high antibody response to HPV16E7 had significant seroreactivity to CT-gp96 fragment. In Western blot analysis, a strong association between anti-E7, anti-NT-gp96 and anti-CT-gp96 reactivity and cervical cancer was obtained using purified recombinant proteins. In adenocarcinoma cases, no significant difference was observed in seroreactivities between normal and patients. Interpretation & conclusions: The evaluation of cervical cancer patients' seroreactivities against three recombinant proteins (rE7, rNT-gp96 and rCT-gp96) showed significantly higher levels of these markers in SCC only, but not in adenocarcinoma and control groups. Also, the usage of both techniques (ELISA and Western blotting) can provide more reliable tools for diagnosis of cervical cancer.