| IntroductionWorldwide, lung cancer is the most common malignancy and the leading cause of cancer mortality. The role of tobacco smoking in the etiology of lung cancer is well established, but only about 15％of smokers develop lung cancer. Why? The answer is the variation in susceptibility between individuals. Studies show that cells have evolved DNA repair capacity (DRC) to maintain the genome integrity. DNA repair is a complicated system. There are several pathways and many enzymes in it. Different pathways depend on different kinds of DNA damage. In mammals, the pathways include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination repair (HRR), and non-homologous end joining (NHEJ). Because cells must face to the attacks from endogenous or exogenous genotoxic chemicals, the extent of damage repaired by DNA repair system is of great importance.Host cell reactivation assay (HCR) is a powerful tool, which allows researchers to measure the capacity of whole DNA repair system. HCR uses a plasmid with a reporter gene chloramphenicol acetyltransferase (CAT). When the reporter gene is damaged by physical or chemical factors, the reporter gene can not be expressed. Undamaged plasmids and damaged plasmids are transfected into the host cells at the same time. Measure the expression of CAT in the two types of host cells using 3Hchloramphenicol and liquid scintillation counter. The ratio of the expression of damaged plasmid and undamaged plasmid is used to show the DNA repair activity. Recently, more and more studies have focused on the single nucleotide polymorphisms (SNPs) of DNA repair genes. Though most of the studies showed there were associations between the SNPs and lung cancer risk, the results were different among various races or histological types. But DRC is a process involved many genes and multiple steps, it is more suitable to be the marker than the SNPs. So this study is of great importance for screening of the high risk subpopulation and the primary prevention of non-small cell lung cancer (NSCLC).ObjectiveTo evaluate the effect of DRC on the risk of NSCLC. To evaluate the interactions of DRC and smoking on the risk of NSCLC.MethodsThere were 107 cases and 117 controls in this case-control study. They were frequently matched according to sex, age (±5) and smoking status. Lymphocytes were isolated from peripheral blood, then stored in -80℃. p-CAT-control plasmids were treated by UVC (254nm) using the dose of 0J/m2 or 800J/m2 respectively. The lymphocytes were thawed and cultured in RPMI1640 medium (supplement of phytohemagglutinin) in batches. The cells were transfected with damaged or undamaged plasmids by DEAE-dextran methods after 72h. Cell lysis was extracted after 44h, stored in -80℃. The cell lysis reacted with 3H-chloramphenicol after being thawed. With appropriate scintillation fluid, the radioactivities of the samples were measured by a liquid scintillation counter. The cpm values were recorded for the cells with undamaged plasmids (control reading) and UV-damaged (repair reading) plasmids. DRC (％)=(damaged plasmid value/undamaged plasmid value)×100％.Pearson's x2 test was used to test the differences in the distributions for qualitative variables. DRC data were analyzed as a continuous variable for the variations between cases and controls. Student's t-test was used to compare DRC by sex, age, smoke status, histological types, pathologic stage, and differentiation. In order to evaluate the OR of DRC on the risk of NSCLC, we divided the subjects into two groups according to the DRC median in controls. In order to evaluate the OR of cumulative smoking on the risk of NSCLC, we divided the subjects into two groups according to the smoking index median in controls. Then we evaluate the OR of DRC combined smoking on NSCLC.Results1. General results about DRC in cases and controlsDRC of controls was higher (9.04％) than that of cases (7.73％) significantly (P＜0.001). DRC of controls was higher than that of cases after the subjects were stratified by sex, age. DRC of males was higher than that of females. DRC of younger subjects was higher than that of older subjects in controls. When the subjects were stratified according smoking status (non-smoker, ever smoker, current smoker), DRC of controls was significantly higher than that of cases in each stratification. And when the cases were stratified according to histological type, pathological stage or the extent of differentiation, DRC in cases was lower comparing to that of controls.2. The effect of DRC and cumulative smoking on the risk of NSCLC and each histological type.(1)The subjects were divided into two groups according to the DRC median of controls, named better DRC and poor DRC. Poor DRC was associated with the risk of NSCLC, OR value was 4.026 (2.211-7.329). OR for adenocarcinoma was 2.949 (1.397-6.225), OR for squamous cell carcinoma was 5.196 (2.144-12.596), and OR for other type was 6.390 (1.381-29.574).The subjects were divided into two groups according to the smoking index median of controls, named heavy smoking and light smoking. Heavy smoking was associated with the risk of NSCLC, OR value was 2.227 (1.284-3.860) for NSCLC, 2.265(1.078-4.758) for adenocarcinoma, 3.195 (1.448-6.864) for squamous cell carcinoma and 3.800(1.019-14.169) for other type.(2)The subjects were divided into four types: better DRC+light smoking group (reference group), better DRC+heavy smoking group, poor DRC+light smoking and poor DRC+heavy smoking group. OR of poor DRC+heavy smoking group was 7.154 (2.960-17.290) for NSCLC, 4.673 (1.659-13.164) for adenocarcinoma, 7.615 (2.032-28.537) for squamous cell carcinoma, and 11.423 (1.376-94.860) for other type. But in poor DRC+light smoking group, only OR for squamous cell carcinoma was statistically significant. The value was 4.091 (1.071-15.621). The results indicated that poor DRC and heavy smoking associated with the risk of NSCLC. And the interactions between DRC and smoking increased the risk of NSCLC.ConclusionPoor DRC is a very important susceptible factor for the risk of NSCLC. The interactions between DRC and cumulative smoking can increase the effect of either factor on the risk of NSCLC. The individuals with poor DRC and heavy smoking are more susceptible to NSCLC. IntroductionDNA double-strand breaks (DSBs) may be the most serious type among all damages that cells have to face everyday. Recently, the studies about DSBs have suggested that histone H2AX is of great importance during DSBs repair process. At the beginning of repair process, H2AX is phosphorylated at Ser139, calledγ-H2AX. Large-scale phosphorylation of H2AX can form nuclear foci (NF), and the number of NF is the same with that of DSBs. H2AX is phosphorylated rapidly after DSBs occur in cells. After theγ-H2AX expression arrives at peak value,γ-H2AX will be dephosphorylated by some important enzymes. So the number of NF reduces. The kinetics of H2AX phosphorylation-dephosphorylation is consistent with DSBs repair process. This suggests that there is some important association betweenγ-H2AX and DSBs repair.The capacity of cells to repair DSBs is optimal, ifγ-H2AX arrives at the peak value more rapidly, or the remaining foci are less. Histone phosphorylation may facilitate the repair factors assembling to and remaining at break sites. The roles ofγ-H2AX in DSBs repair indicate that it is of great importance in remaining the integrity of genomic DNA. Previous studies aboutγ-H2AX usually used immunofluorescence and computer image techniques to measure the expression ofγ-H2AX in individual cells. Though the technique is very classical and accurate, it involves small part of cells and is time-consuming. So it isn't suitable for people-based studies. -ObjectiveTo quantitatively evaluate the expression ofγ-H2AX in A549 cell line after it is treated by exogenous factors, such as x-ray and cisplatin using flow cytometry (FCM). To quantitatively reflect the kinetics ofγ-H2AX phosphorylation-dephosphorylation. To provide experimental basis for using thisγ-H2AX detection technique in future people-based epidemiological studies.Methodγ-H2AX was measured using FCM and H2AX kit for FCM (Upstate company). Culture medium was changed after subculture 72h. The cells were irradiated by x-ray. Energy of irradiation source was 6MV, the field was 40cm×40cm, the distance between source and object was 100cm, and the rate was 200cGy/min. The dose of irradiation was 2Gy, 5Gy, 10Gy, 15Gy, 20Gy. FCM analysis was performed after incubation for 0.5h, 1h, 2h, 4h, 6h, and 24h. The powder of cisplatin was dissolved in, and the concentration was 1mg/ml. The final concentrations in DMEM medium were 2μg/ml,4μg/ml,6μg/ml,8μg/ml and 10μg/ml, respectively, cell medium was replaced by these DMEM mediums. FCM analysis was performed after incubation for 0.5h, 1h, 2h, 4h, 6h, and 24h.Result1. After cells were treated by x-ray, theγ-H2AX expression was highest at 0.5h in all dose groups. It indicated that theγ-H2AX expression might arrive at peak value within 0.5h. Then theγ-H2AX expression decreased, especially in 2Gy and 5Gy group. Theγ-H2AX expression at 1h was 30％of the level in 0.5h. The decreasing rate was slower in 10Gy group and 15Gy group. Then there seemed to be a platform in 15Gy group between 1h to 2h. But theγ-H2AX expression in other groups continued to decrease. At 24h, theγ-H2AX expression level in 2Gy group was lower than control group, that in 15Gy group and 20Gy gourp was very close to control group, but that in 5Gy and 10Gy was appreciably higher than control group. 2. The peak value ofγ-H2AX expression was between 2h-4h in all dose groups after the cells are treated by cisplatin. The time of peak expression was later than that in x-ray treated groups. Theγ-H2AX expression fluctuated slightly between 0.5h to 24h in 2μg/ml and 4μg/ml dose groups. When the cells were treated with 6μg/ml cisplatin, theγ-H2AX expression increased slowly. The peak value occured at 2h, then decreased slowly. When the cells were incubated for 6h, theγ-H2AX expression was close to controls. While there were highγ-H2AX expression at 24h in 8μg/ml group and 10μg/ml group.ConclusionDetection ofγH2AX with FCM is a sensitive indicator of DSBs repair in cells which are exposed to exogenous factors, such as x-ray and cisplatin. And the technique can show kinetics associated with DSBs repair in large number of cells. The technique has potential in clinical application in measurement of individual sensitivity to treatment, because it is simple, rapid and accurate. IntroductionBreast cancer is a common disease in Westem females, with a high rate incidence from 1 in 8 to 1 in 12 in the Caucasians. In the last decade, there have been many important advances in understanding genetic susceptibility. In 1990, using the newly DNA sequence polymorphisms, the genetic susceptibility of the breast cancer was dispelled by demonstrating of genetic linkage in breast cancer families. First-degree relatives of breast cancer patients have a 2-fold increase in risk over the general population. In principle, the remaining 80-85％of familial risk might have a genetic or environmental origin. Unfortunately, the evidence from studies of breast cancer in twins, tumor incidence in the contralateral breast of an affected individual and the pattern of inheritance in families suggests that genetic factors predominate. In contrast, the similar studies suggests, BRCA-Iike genes are unlikely to account for much of the risk, which might be explained better by a model that describes the combined action of several factors, each with an individually mild effect. For breast cancer, the greater part of inherited predisposition may due to the effects of combinations of genetic variants at several of different loci. However, the contribution of low-penetrance genetic variants or polymorphisms to the risk of sporadic cancer development remains unclear. Current efforts were being focused on identifying and characterizing genetic variants that predispose female to breast cancer and considring to identify additional high- and low-penetrance susceptibility genes. Recently, one of few report from large-scale association study using high throughput single nucleotide polymorphisms (SNPs) genotyping at UHN to collect German cases and controls and＞25,000 SNPs located within 16,000 genes were conducted, so as to identify genetic variations that influence breast cancer risk. Three SNPs of intercellular adhesion molecule 5 (ICAM5) variants on chromosome 19p13.2-19p13.3 were identified to be associated with increasing risk increase of breast cancer.ObjectiveTo explore the association between SNPs in ICAM5 gene and breast cancer risk. To exprlore the effect of interactions between SNPs in ICAM5 and BRCA1 on breast cancer risk.MethodOne thousand and thirty-four breast cancer cases and one thousand and ninty-one controls in our study. After isolation DNA from peripheral blood, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF) to determine the genotype of every SNPs. We used x2 test and logistic regression model to analyze the associations between SNPs in ICAM5 gene loci and the risk of breast cancer in all subjects. We compared the variations in the risk between the subjects with BRCA1 and the subjects without it. Logistic regression model was used to measure the effect of SNPs in ICAM5 gene on breast cancer risk and the effect of interaction between ICAM5 gene and BRCA1 on the breast cancer risk, after adjusting the confounding factors.Resultrs1056538 with C allele might have some prevention effect on women breast cancer risk (ORCT=0.3423, 95％CI: 0.2351-0.4982; ORCC=0.5943, 95％CI: 0.3899-0.9058). While women whose genotype was rs2228615AG or rs281439GG would have higher breast cancer risk (ORrs2228615AG=2.6771, 95％CI: 1.4938-4.7976; ORrs281439GG=1.9845, 95％CI: 1.0287-3.8285). For women with BRCAI mutation, compared with the results of all subjects, rs1056538 with C allele might have greater prevention effect (ORCT=0.2797, 95％CI: 0.1984-0.3321; ORCC=0.4983, 95％CI: 0.3942-0.7211), rs222861 with G allele would have higher breast cancer risk (ORAG=3.3369, 95％CI: 1.9195-5.8009; ORGG=2.0289, 95％CI: 1.1704-3.5170), but rs281439GG genotype had little diffenence in the effect on the risk.ConclusionThere is an association between SNPs in ICAM5 gene and breast cancer risk. The interactions between SNPs in ICAM5 gene and BRCA1 have some effect on breast cancer risk. |
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