PAH-DNA Adduct, Polymorphisms Of P53, P73, HER-2 And Colorectal Cancer Risk

Backgrounds and ObjectivesColorectal cancer,including colon and rectum cancers,is a major cause of cancer-related incidence and mortality.At present,colorectal cancer,breast cancer and lung cancer are considered as the three of the most prevalent malignant tumors worldwide.The incidence of colorectal cancer was lower in our country compared with those in western countries.However,during the past decades,the incidence and mortality of eolorectal cancer have increased significantly with the remarkable changes of life style and environment which induced by rapid development of socio-economic. Now,colorectal cancer is considered as one of the diseases which impair the quality of life severely in our country.Most human cancers,including colorectal cancer,have a multifactorial etiology involving complex interplay of environmental exposures and genetic susceptibility. Because of lacking direct evidence of identification and quantification of environmental factors by traditional epidemiology,the association between environmental exposures and diseases were not explained and the results were not consistent.With the development of molecular biology,DNA adduct can be used as interal exposure marker or effect marker,so the DNA adduct has been applied gradually in the estimation of cancer risk.Among a variety of DNA adducts,PAH-DNA adduct has been researched extensively and many studies have shown that PAH-DNA adduct is correlated with a few of malignant tumors.Besides,many researches suggested that the susceptibility of colorectal cancer is associated with different kinds of genetic alterations such as mutation in oncogenes and tumor suppressor genes.The p53 gene is a crucial tumor suppressor taking effect on DNA repair,cell cycle arrest and apoptosis in some cases via its stress way,which is essential for genomic stability,and plays an important role in preventing tumor formation.The p73 gene,a p53 homologue with respect to both sequence and DNA-binding domain,encodes a protein which activates several p53-responsive genes involved in cell cycle control,DNA repair and apoptosis,and inhibits cell growth in a p53-like manner by inducing apoptosis.In addition, amplification and/or overexpression of HER-2 have been observed in various cancers, and it is suggested that polymorphism of HRE-2 may also account for the genetic difference in colorectal cancer susceptibility.A case-control study was conducted in Jiashan County to describe the level of PAH-DNA adduct and the frequency distribution of p53 Intron3 16bp duplication,Exon4 Arg72Pro,p73 G4C14-A4T14 as well as HER-2 Ile655Val polymorphism among natural Chinese population,and to explore the association of colorectal cancer risk with PAH-DNA adduct,the above single polymorphism,environment-DNA adduct interaction between environmental factors(cigarette smoking and alcohol drinking) and PAH-DNA adduct,as well as gene-DNA adduct interaction between the above polymorphisms and PAH-DNA adduct.Material and MethodsThis case-control study recruited eligible new patients with histologically confirmed colorectal cancer diagnosed during 1^st Jan 2005 to 31^th Jul 2008 as cases. Although there were no restrictions on patients' age,gender or tumor stage,only those patients who were free of metastases or other cancers were included in our study. Simultaneously,controls those did not have a history of cancer were individually matched with cases by 5-year categories,gender and residence.All the participants were ethnic Han Chinese and residents in Jiashan County.As a result,295 case-control pairs were formed,After informed consent,all subjects were face-to-face interviewed with a structured questionnaire mainly including demographic characteristics,individual lifestyle(cigarette smoking,alcohol drinking,etc) and family history of cancers by trained interviewers.Meanwhile,a 5 ml venous blood sample was collected from each subject with the permission and then the genomic DNA was extracted from peripheral blood sample by modified salting-out procedure.For determination of the genetic polymorphisms of p53 Exon4 Arg72Pro and HER-2 Ile655Val,polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique was performed.The direct electrophoresis after PCR amplification was applied for the genotyping of p53 Intron3 16bp duplication and p73 G4C14-A4T14.The PAH-DNA adduct was detected by a competitive enzyme linked immunosorbent assay(ELISA).After verificatied and coded,the questionnaires were inputted double times using Epidata software to set up database.PAH-DNA adduct level with non-normalized distribution was expressed by median and interquartile range and the differences were examined by nonparametric tests.McNemar test and Wilcoxon signed rank test were applied to compare the differences in categorical variables and continuous variables between cases and controls respectively.The Hardy-Weinberg equilibrium test in controls was finished byχ² goodness-of-fit test.Conditional logistic regression was used to calculate the odds ratios(ORs) with 95%confidence intervals(95%CIs).Interactions were formally assessed by examining departures from multiplicity by comparing models with and without the interaction term using the likelihood ratio test(LRT).All statistical analysis was finished in Excel 2003 and SPSS 13.0 for windows.All tests of statistical significance(α=0.05) were two-sided.Results1.Distribution of PAH-DNA adduet in subjectsThe median of PAH-DNA adduct level was 9.12/10⁷ nucleotides(4.00-25.81/10⁷ nucleotides) of 565 subjects whose blood sample was enough for detection of PAH-DNA adduct.The level of PAH-DNA adduct in male was significantly higher than that in female.However,differences of adduct level between age,BMI,family history of cancer were not significant(p>0.05).Similarly,there were no significant differences of adduct level between cigarette smoking,age began smoking,smoking years, cigarettes per day,alcohol drinking,age began drinking as well as drinking years (p>0.05). 2.PAH-DNA adduct and colorectal cancer riskCompared with the nondetected group,the group with higher level of PAH-DNA adduct than the median of controls showed a trend of increased risk of colorectal cancer, and the OR was 1.67(95%Cl:0.97-2.89).In addition,the joint actions between age began drinking(<30 years old),drinking years(≥30 years) and PAH-DNA adduct had an increasing risk of colorectal cancer,and the ORs were 4.48(95%CI:1.01-19.88) and 4.41(95%CI:1.16-24.46),respectively.3.Polymorphisms of p53,p73,HER-2 and colorectal cancer riskWe did not obsevered a significant association between any single p5316bp duplication,Exon4 Arg72Pro,p73 G4C14-A4T14,HER-2 Ile655Val polymorphism and colorectal cancer risk.However,the joint actions between p73 GC/AT&AT/AT genotypes,HER-2 Ile/Val&Val/Val genotypes and PAH-DNA adduct made colorectal cancer risk increased significantly,with the ORs were 1.94(95%CI:1.09-3.91) and 2.22 (95%CI:1.10-4.48) respectively.Additionally,the carriers with three or more high-risk genotypes and PAH-DNA adduct detected showed three-fold increased risk(OR=2.98, 95%CI:1.07-8.31) of colorectal cancer compared with low-risk genotypes carriers with nondetected DNA adduct.ConclusionsThe level of PAH-DNA adduct in male was significantly higher than that in female. Age began drinking(<30 years old) and drinking years(≥30 years) could aggravate the risk of colorectal cancer induced by PAH-DNA adduct.Additionally,the high-risk genotypes of p53,p73 and HER-2 carriers with PAH-DNA adduct detected show three-fold increased risk of colorectal cancer compared with low-risk genotypes carriers with nondetected DNA adduct.In a word,it is feasible to estimate the colorectal cancer risk by PAH-DNA adduct combined with the polymorphisms of p53,p73 and HER-2.