1. Functional Polymorphisms Of JWA Gene Are Associated With Risk Of Bladder Cancer 2. The Drosophila Homolog Of JWA Is Required For Ethanol Tolerance

Bladder cancer is the most common malignant tumor among urinary-system.Recently,there is an increasing trend in incidence of bladder cancer world wide,and this trend is more significant in China. The development of bladder cancer is a multiple factors and multiple phases process,and is the result of enviromental risk factors and genetic factors together.Epidemiological investigations show that exposure to enviromental risk factors is vital to the etiology of bladder cancer. Although many people are exposed to these risk factors,only a fraction of exposed individuals develop bladder cancer,suggesting a variation in individual susceptibility to bladder carcinogenesis,suggesting that individual susceptibility to exposure-related carcinogenesis may vary.JWA(GenBank:AF070523.1,1998),a newly identified ATRA-responsive gene,was initially cloned from primary human tracheal bronchial epithelial cells by Zhou.There is an increasing amount of data to indicate that JWA is a structurally novel microtubule-associated protein (MAP),induced by various agents such as TPA,ATRA,4HPR or As₂O₃, as well as stress conditions including heat shock and cold shock,and initiation of cell differentiation in leukemia cells.Several JWA homologues(e.g.,ARL-6,GTRAP3-18,addicsin,and JM4)were identified in recent years.ARL-6 is a member of the ADP-ribosylation factor-like family,which shows 93%homology with JWA and its functions mainly referred to DNA damage and repair.Our recently studies found that the JWA gene served as an environmental stress sensor to protect cells against DNA damage,which was associated with reactive oxygen species;JWA might be a upstream regulatory factor of XRCC1 and PARP1,and involved in the damage site repair and gap filling;it play a role in XRCC1-PARP1 pathway.In addition,we found that JWA is involved in MAPK signaling pathway,and associated with cell migration and movement,which is the prerequisite for tumor cell infiltrating and metastatic.These results intensively suggest that JWA might be a potential tumor suppressor and an important signaling molecule involved in tumor cell differentiation.In this study,we identified four polymorphisms of JWA gene by PCR-SSCP method.In a bladder cancer case-control study,we analyzed the association between JWA polymorphisms and risk of bladder cancer. We also detected the JWA protein levels in bladder or bladder cancer tissues.Finally,we studied the transcription factor which may influenced by -76G＞C variation.1.Identification of JWA polymorphisms and functional analysis of -76G＞C variationIn this study 4 SNP were identified in the Chinese population by PCR-SSCP.It was not possible to identify all reported common exonic SNP;however the presence of two SNP(723T＞G;rs7038 and 454C＞A, rs10489)were confirmed and two additional novel SNP(-76G＞C and 380G＞A)identified in JWA.The report gene assay showed that the variant -76C allele had a more-than 4-fold and 12-fold loss of the transcript activity in untreated and B(a)P treated cells,respectively, compared with the level of the -76G allele.To test whether JWA gene would response to B(a)P treatment in translation level,we detected the JWA protein level in untreated of B(a)P treated NIH-3T3 cells.We found that JWA protein significantly increased after exposure to B(a)P.These results showed that -76C variation made JWA gene an significantly decreasing response to the ROS produced by B(a)P.We also detected the genotypes and allele frequencies of the -76G＞C polymorphisms in different ethnic groups,and found an ethnic difference among four ethnic populations.2.Association between JWA polymorphisms and risk of bladder cancerIn bladder cancer case-control study,we found a statistically significantly increased risk of bladder cancer was associated with the -76G＞C variation or 454C＞A,however,the 723T＞G variation was associated with a significantly decreased risk of bladder cancer.Linkage disequilibrium analysis indicated that the -76G＞C and 454C＞A polymorphisms were in linkage disequilibrium,suggesting that there existed haplotypes that may exert an enhanced effect on the risk.When the haplotype genotypes were regrouped by the number of variant alleles and the GC/GC genotype 0 variant allele was used as the reference,the risk was significantly increased as the number of variant alleles rose. Further stratification analysis showed that the increased risk appeared to be more pronounced among older(＞65 years)individuals,male,ever smokers,and ever drinkers.Whereas -76G＞C variation may be functional, and may influence the response level of JWA gene to oxidative stress,we detected the gene-environment interaction between -76G＞C and smoking/drink.The result suggested that smokers of drinkers with -76CC genotype had a significant increased risk of bladder cancer;however, there was no statistical evidence for the interaction,which may be due to limited study power.To test whether -76G＞C variation would influenced the JWA protein level in tissues,we detected the JWA protein levels in bladder or bladder cancer tissues with -76GG or -76GC genotype by immunohistochemistry.We found that the JWA protein level in -76GG genotype tissues was much higher than those in -76GC genotype tissues. All these results showed that JWA may be a potential tumor suppressor, and JWA SNPs were associated with susceptibility to bladder cancer.3.Identification of transcription factor influenced by JWA -76G＞C variationFirstly,we searched the possible transcription factors which might bind with the sequence including -76G＞C polymorphism by Alibaba 2 software,and the result showed that changed the former C/EBPαbinding site into C/EBPβbinding site.The further electrophoretic mobility shift assays(EMSA)confirmed that some nucleoprotein bond with -76GG probe in H₂O₂ or B(a)P treated group.After Southwestern assay,we found the molecular weight of the nucleoprotein was about 43 kD,and this is consistent with C/EBPα.So we supposed that the transcription factor influenced by -76G＞C variation was C/EBPα.4.To construct normal cell line with JWA -76GC or -76CC genotypeTo further analyze the function of JWA -76G＞C variation in cell level,we decided to construct a normal cell line with JWA -76GC or -76CC genotype.However,we had not get any normal cell line with JWA -76GC or -76CC genotype,even after times of improvement in experiment technique or change other kinds of normal cell line.Taken together,we identified four SNPs of JWA gene:-76G＞C, 380G＞A,454C＞A,and 723T＞G.The report gene assay showed that the variant -76C allele made JWA gene an significantly decreasing response to the ROS produced by B(a)P,and the mechanism may be the variation weakened binding ability of C/EBPαwith JWA promoter.In bladder cancer case-control study,we found a statistically significantly increased risk of bladder cancer was associated with the -76G＞C variation or 454C＞A,however,the 723T＞G variation was associated with a significantly decreased risk of bladder cancer.We also found that -76G＞C variation could influence the JWA protein level in tissues.In addition, because of several kinds of reason,we failed to construct normal cell line with -76GC or -76CC genotype. Alcohol abuse poses a serious public health problem,and its repeated use can produce tolerance,leading to dependence and addiction. However,the mechanisms for alcohol tolerance and addiction are not fully understood.Drosophilae have been employed as a suitable model to study the molecular mechanisms of ethanol tolerance.JWA,a newly identified microtubule-binding protein,regulates cell stress, transportation of intracellular excitatory amino acids,and involves in MAPK signal pathway.Addicsin,the alias of JWA,has been considered involving in the development of morphine tolerance and dependence. This study was designed to examine whether the JWA participate in the mechanisms of ethanol tolerance in Drosophila.We found the JWA homologous gene in Drosophila,CG10373 (dJWA),and constructed the anti-dJWA and cDNA-dJWA fly strains by microinjection.Real-time PCR was used to measure the dJWA levels in different kinds of fly strains.Rapid tolerance experiments including inebriator exposure and recovering assay. JWA gene is evolutionary conservation with 41.4%of the amino acids and 53.6%of the DNA bases are identical between the human and Drosophila melanogaster.In inebriation tests,the wll18 flies acquired ethanol tolerance after several times of exposure,and so did the cDNA-dJWA flies.However,the anti-dJWA flies,the tolerance to alcohol exposure disappeared.Here we show that the Drosophila dJWA gene has a critical role in ethanol tolerance.Flies with a dJWA knockdown did not acquire ethanol tolerance after repeated exposures to ethanol.Base on the previous studies,JWA might be a vital functional molecule in the mechanisms of ethanol tolerance and drug addiction.Our results present a new direction for alcohol tolerance and addiction researching.