Study On P53 Mutations And Polymorphisms In Human Papillomavirus-associated Esophageal Cancer

Esophageal cancer (EC) is one of the most common cancers worldwide with broad variation in its incidence among different countries, different ethnic or sex groups. In western countries, heavy cigarette smoking and alcohol intake are thought to be major risk factors.Whereas, in some regions of Asia, exposure to dietary carcinogens and nitrosamine are believed to be the major etiological factors. In 1982, Syrjanen et al firstly reported an involvement of human papillomavirus (HPV) in the development of both benign and malignant squamous cell carcinomas (SCC) of the oesophagus. Since then, a substantial amount of literatures have accumulated on this subject. To date, many different HPV detection methods have been used, with HPV being detected in 0%-70% cases in different areas. However, generally, the highest HPV prevalence in esophageal cancer was found in high-risk areas, whilst the lowest was found in low-risk areas. Many studies have been done on SCC. In addition, evidence derived from large-scale serological studies, animal experiments, and in vitro studies, was discussed in the light of the highly variable geographical incidencerates of esophageal cancer worldwide. It may be that the multi-factorial aetiology of esophageal cancer differs greatly between those geographical areas with the low risk and those with the high risk for this disease. Oncogenic HPV types seem to play an important causal role, particularly in high-risk areas. Anyang area in Henan province is one of the high incidence areas of esophageal cancer in China, even in the world. For several decades, much work on the etiology and carcinogenesis of esophageal cancer has been conducted. In recent years, some studies have been done on the prevalence of HPV in the tissues of esophageal cancer in Anyang area, the conclusions were not consistent.Therefore, it is still questionable whether HPV is the major cause of esophageal cancer in this high-risk area.p53 gene is a putative tumor suppressor gene, which has been implicated in the control of cell cycle, DN A repair, cell differentiation and programmed cell death. The mutation of p53 gene is an important pathway that causes p53 protein to lose its function; high-risk HPV E6 protein can bind to p53 protein and lead to the degradation of p53 protein through the ubiquitin pathway. In recent years, study shows that at least some high-risk HPV types can inactivate p53 suppressor function and thus can be considered alternative pathways to chromosomal instability, uncontrolled proliferation, and malignant transformation in precursors of cancers of the oral cavity (OC) and oro-pharynx (OP). Although some studies have evaluated HPV presence and p53 statues in esophageal cancer, the combined evaluation of p53 gene and marker of p53 function induced by HPV infection has never been performed.Exon4 of p53 gene can control the growth and apoptosis of cell. The ubiquitin-dependent proteolytic pathway plays a major role in selective protein deregulation. E6 oncoprotein of high-risk HPV types uses this cellular proteolytic system to target p53 protein. E6 oncoprotein binds to a cellular protein of E6-AP (E6-associated protein), and the E6-E6-AP complex interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. At present, in respect to the function of p53 polymorphism in HPV-associated esophageal cancer, it remains unclear. Some studies implied that the frequent loss of proline allele on codon 72 ofexon 4 may play a role in HPV-associated esophageal cancer.In order to explore the prevalence of HPV and p53 mutations and polymorphisms in cacinoma tissuses of local residents in Anyang area and to evaluate their possible roles and interaction in the carcinogenesis, here we present the following experiments. Some usefull findings may be provide scientific basis to prevent esophageal cancer.Methods1. Collection of specimensA portion of the biopsy used for histological diagnosis was taken from each case by the pathologist in Anyang Tumor Hospital. Each specimen was kept in a freeze tube, labelled and stored at -80°C.2. DNA extractionDNA will be extracted from the tissues according to NaCl method and was resuspensed in TE buffer and stored at 4°C.3. HPV detectionFirstly, P-globin PCR analysis was performed to confirm the presence of human DNA in specimens. Then HPV DNA was detected by PCR method with degenerate primer and specific primer to HPV 16 E6.4. Polymorphisms on codon 72 of p53PCR-RFLP(Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method was used to analyze the polymorphisms on codon 72. Briefly, exon 4 was amplified by PCR, then Bst UI was used to do RFLP fingerprint.5. p53 mutations analysisExon5-9 of the p53 gene was analyzed for the presence of mutations. 3 pairs of oligonucleotide primers were used for Touchdown PCR in 3 separate reactions to produce 3 fragments:exon5-6(467bp),exon7(237bp) and exon8-9(445bp). Mutations were screened by Single Strand Conformation Polymorphism (SSCP). For samples with positive or doubtful results of SSCP, Touchdown PCR wasrepeated to generate the template for DNA direct sequencing. Results1. The PCR result showed that the positivity of HPV 16 in esophageal carcinoma tissues was 49.09%, and 15.00%, 16.00% respectively in the normal mucosa and the controls in Zhengzhou. There is a significant difference among them.2. The frequency of Arg/Arg genotype on codon 72 of p53 was higher in cases (42.73%) than in controls (17.76%).There is a significant difference between them.3. The frequency of Arg/Arg homozygosite was 55.56% in HPV DNA positive groups, compared with 30.36% in HPV DNA negative group. There is a significant difference between them.4. The rate of p53 gene mutations was 49.09% in esophageal cancer cases; the rate of mutations in exon5-6, exon7, exon8-9 were 19.09%, 27.27% and 17.24% respectively.5. There was no association between p53 mutations and sex, age and drinking habits; There was an association between p53 mutations and lymph node metastasis and smoking.6. The rate of p53 gene mutation was 40.74% in HPV 16 negative groups and 57.14% in HPV16 positive groups.There was no association between HPV16 status and p53 mutations.Conclusions1. There is high rate of HPV DNA detection for cases of Anyang area. The statistical analysis shows distinctly significant difference between cases and controls, the results indicate that high-risk HPV plays an important role in the development of esophageal cancer.2. The frequency of p53 Arg/Arg genotype is higher in cases than in controls.There is distinctly significant difference between them. The result suggests that p53 Arg/Arg genotype is one of high risk genetic factors for cases in Anyang area.3. It is noteworthy that the distribution of Arg/Arg homozygote on condon 72 ofp53 in HPV positive cancers is significantly higher than HPV negative cancers. The result shows that Arg/Arg homozygote on condon72 of p53 is one of the genetic factors for HPV-associated esophageal cancer in Anyang area.4. There is a significant difference between p53 mutations and lymph node metastasis. This result may predict that the cases could be deteriorated after the operation. Follow-up investigation of our results should shed light on how to determine and possibly alter the potential malignance of esophageal cancer.5. There is no association between HPV16 status and p53 mutations. The result shows that p53 gene mutations may play an important role in the mechanism of esophageal cancer and support the relationship between p53 mutations and HPV infection not to be mutually exclusive.