Studying DNA Repair Capacity And Checkpoint Gene Protein (ATM Protein) Of Lung Cancer Patients And Breast Cancer Patients

Tumor is resulted from the interaction between environmental factors and human inherent factors (genetic factors) . So it is a disease relate to environment. Some environment factors, such as air pollution water pollution smoking diet occupation and human life style are associated with the development of cancer. Moreover, the inherent factors are very important for the development of tumors. Therefore, American government started Environmental Genomics Project (EGP) to study the function of a series of environmental response genes which include DNA repair genes. Fig 1 shows the role of DNA repair in carcinogenesis. DNA repair is a defense system to protect the integrity of the genome. Deficiencies in this system can result in the development of cancer. Therefore, DNA repair capacity and of its effect on cancer susceptibility in humans is an important area of environmental tumor epidemiological investigation. As a consequence of DNA damage induced by environmental carcinogens, a number of sophisticated sensing and transduction systems are initiated, and the damage signal is conveyed simultaneously to multiple effectors. The ataxia telangiectasia mutated (ATM) protein plays a central role in these processes. This multifunctional protein kinase determines the intricate array of cellular reactions to double strand breaks (DSBs) by phosphorylating numerous substrates, which are involved in specific signaling pathway. Both of lung cancer and breast cancer are the cancers related to the environment. The aim of this research work is to study the difference of DNA repair capacity between cancer patients and healthy controls, toanalyze the relationship between DNA repair capacity and cancer susceptibility and to observe whether ATM protein expression levels of lung cancer patients and breast cancer patients decrease, and explore the relationship between DNA repair capacity and ATM protein expression levels, which could provide molecular biological theoretical evidence for the prevention of lung cancer and breast cancer. This work is divided into the following parts: First-phase research: Method creation(1) Detecting DNA repair capacity of human lymphocytes using comet assay.(2) Comparing the UVC challenge test with bleomycin challenge test for detection of DNA repair capacity in cancer patients.Central research:( 3) Studying DNA repair capacity and ATM protein expression level in breast cancer patients(4) Studying DNA repair capacity and ATM protein expression level in lung cancer patients(BER);Nucleotide excision repair (NER);Homologus recombinational repair(HRR) Non-homologus end-joining (NHEJ) and Mismatch repair (MMR). So far, five kinds of techniques have been used to estimate DNA repair capacity. However, it is necessary to develop a reliable, rapid and sensitive assay for the tumor molecular epidemiological investigation. The comet assay or single cell gel eletrophoresis is considered as a simple, rapid and sensitive assay for detecting several types of DNA damage. In this study, UVC as a mutagen, novobiocin (NOV) and aphidicolin (APC) as the inhibitors of DNA repair were used to study the detection of DNA repair capacity. NOV and APC act at the incision and ligation steps in DNA repair process, respectively. Heparinized venous blood was collected from 12 young healthy donors(6 males and 6 females, 25-26 years old), then the lymphocytes were isolated from whole blood. The lymphocytes of each donor were divided into three parts: (1) UVC group( 2 )UVC plus APC group;(3 )UVC plus NOV group. DNA single strand breaks were detected with comet assay in unirradiated cells and UVC-irradiated cells incubated for: 30, 60, 90, 120, 180 and 240min. DNA repair rate (DRR) was calculated and served as an indicator of DNA repair capacity. The result indicated that the maximum MTLs of 11 donors appeared at incubation 90min. Only the maximum MTL of donor 5 presented at incubation 120 min., which was 2.96um adjacent to MTL value (2.93um) at incubation 90min. At incubation 240 min, the average MTL was 2.43 urn, which was significantly lower than that at 90min and adjacent to the level (1.93 urn) of pre-exposure to UVC. MTL at incubation 90 min of UVC plus NOV group was 3.16 um which was significantly lower than that (4.77 urn) of UVC group(i><0.01). There was no difference between the UVC plus APC group and UVC group. MTL at incubation 240 min of UVC plus APC group was 3.43 um which was significantly higher than that (2.43 um) of UVC group (P<0.01) . It was found that average DRRs of UVC plus NOV or APC groups were 52.98%and 39.57%, respectively , which were significantly lower than that (81.84 %) of UV group(P<0.01) . In this experiment, the MTL values at incubation 90 min after UVC exposure in UVC plus NOV group were significantly lower than those in UVC group. The MTL values at incubation 240 min after UVC exposure in UVC plus APC groupwere significantly higher than those in UVC group. The results indicated that NOV inhibited the incision process of NER and APC inhibited the ligation process of NER. The DRR values of both UVC plus NOV and UVC plus APC groups were significantly lower than that of UVC group, which exhibited that the DRR value could efficiently reflect the decreased repair capacity induced by either the inhibition on incision process or ligation process. Our study displayed that comet assay can be utilized to assess the human DNA repair capacity by observing kinetic change of DNA strand breaks and calculating DRR.Part II Detecting DNA repair capacity of peripheral lymphocytes from cancer patients using UVC challenge test and Bleomycin challenge testThe objective of this study was to evaluate DNA repair capacity of cancer patients using BLM challenge test and UVC challenge test. The mechanism of UVC action differs from the mechanism of BLM action. DNA damage induced by UVC is repaired through NER pathway, but DNA double strand breaks and single strand breaks produced by BLM (radiometric agent) is repaired through HRR, NHEJ and BER pathways. The human peripheral lymphocytes were collected from 33 patients with different kinds of cancers and 33 controls. The lymphocytes of each subject were divided into two groups: (1) hi bleomycin challenge test, the lymphocytes were treated with bleomycin (20 u-g-mT1) for 30 min, and repaired for 15 min. The DNA damage before and after bleomycin exposure was detected with comet assay to assess DNA repair capacity. (2) In UVC challenge test, the lymphocytes were exposed to UVC (254 nm) at the dose of 1.5 jm'2. DNA damage of lymphocytes was measured before UVC exposure and at 90^ 240 min after UVC exposure using comet assay, then DNA repair percentage (DRP) was calculated. The results of this study indicated that the average DNA repair percentages of cancer patients were 75.63±3.11%and 68.98±4.19%calculated with tail length and tail moment in BLM challenge test, respectively , which were significantly lower than those ( 91.11±1.09%and88.19±1.71%) of controls (P<0.01);the mean DRPs of cancer patients were 49.19±3.47%and 58.27±3.64%calculated with tail length and tail moment in UVC test, respectively, which also were significantly lower than those (77.52±2.06%and 83.12±2.36%) of controls (i)<0.01) . The good correlation between the DRPs (%) drawn with tail length and tail moment in bleomycin test or between the DRPs (%) drawn with mean tail length and mean tail moment in UVC challenge test was foud (P<0.05) .The DNA repair capacity measured with BLM challenge test and UVC challenge test in 33 cancer patients was significantly lower than that in controls. The results suggest the low DNA repair capacity may be an important cancer sensitivity factor. In this experiment, the results of DNA repair capacity levels measured with both of UVC test and BLM test were very similar, but there was a little difference between UVC test and BLM test, that is, the DNA repair capacity level for a few of individuals in UVC test was different from that in BLM test. For this difference, it is possible that individual has different susceptibility to BLM and UVC or there are different DNA repair pathways in UVC test and BLM test. We suggest that two or more different mutagen challenge tests should be used to detect human DNA repair capacity in molecular epidemiological study. The results obtained from various tests will be more objective than those of single test.Part III Studying the multiple DNA repair capacity and ATM protein expression of the peripheral lymphocytes in breast cancer patientsThe aim of this research work was to investigate whether ATM protein expression and DNA repair capacity decrease and the correlation between ATM protein expression and DNA repair capacity in breast cancer patients. Blood samples were collected from 25 female breast cancer patients and 25 female controls. The controls were matched with breast cancer patients on the basis of age, sex and smoking. All patients had not received any primary radio-therapy and chemotherapybefore collecting blood. Some lymphocytes were divided it into two parts that were used in UVC challenge test and BLM challenge test to measure DNA repair capacity, respectively. The detecting methods were the same as Part II using comet assay. The protein was extracted from the other lymphocytes and utilized in western blot test to assess the ATM protein expression level of each subject. In BLM challenge test, while the TL served as a calculating indicator, the DRPs of 5 patients in 25 breast cancer patients were higher than 60%. However, the DRPs of 21 controls were higher than 60%. The average DRPs (40.84%) of cancer patients was significant lower than that(77.09%) of controls (P<0.01) . When TM served as the indicator, the DRPs of 7 patients were higher than 60%, but the DRPs of 21 controls were higher than 60%. Also the average DRPs (41.33%) of cancer patients was significantly lower than that(77.11%) of controls (P<0.01) . In UVC challenge test, the average DRPs for both MTL and MTM in control group were significantly higher than those in patient group(P<0.01). Furthermore, the mean ATM protein expression level was 0.585 in breast cancer patient group and the mean ATM protein expression level was 1.561 in control group. The difference of the mean ATM protein expression level between breast cancer patients and controls was very significant (P<0.01) . When the correlation between ATM protein expression and DRPs measured by two challenge assays was analyzed, only there was a good correlation between ATM protein expression and DRPs measured by BLM challenge assay in patient group, the correlation coefficients were 0.624 (TL) and 0.629 (TM) (P<0.01), respectively. The results of DNA repair capacity in breast cancer patients were similar to those in Part II. The DNA repair capacity measured by either BLM challenge assay or UVC challenge assay in breast cancer patients was significantly lower than that in controls. So the results of our investigation supported the results reported by Ramos's. The reduced DNA repair capacity may be a susceptibility factor for breast cancer. Meanwhile, it was found that ATM protein expression level in breast cancer patients was significantly lower than that in controls (P<0.01) and that there was a good correlation between ATM protein expression level and DRPs measured by BLM challenge test, but no good correlation between ATM protein expression level and DRPs measured by UVC challenge testappeared in cancer group. We suppose the difference of the correlation between ATM protein expression and DRPs measured by two challenge tests may be related to the different mechanisms of two challenge tests, and the difference may be related to the role of ATM protein in DNA repair system. Considering the above reasons, the role of ATM may be a risk factor for breast cancer.Part IV Investigating DNA repair capacity and ATM protein expression in lung cancer patientsThe objective of this investigation was (1) whether the DNA repair capacity of lung cancer patients decreases;(2)whether there is inter-individual difference of repairing the damage induced by two different mutagens in lung cancer patients;(3) whether ATM protein expression decrease and there is a correlation between ATM protein expression and DNA repair capacity in lung cancer patients. Each lymphocyte sample collected from 36 lung cancer patients and 36 controls was divided into three parts. Two parts of lymphocytes were tested by BLM challenge test and UVC challenge test for assessing the DNA repair capacity of lung cancer patients and controls, respectively. The ATM protein extracted from the third part of lymphocytes was detected by western blot The results of both UVC challenge test and BLM challenge test showed the DNA repair capacity of lung cancer patients was significantly lower than that of controls CP<0.01), and the inter-individual difference for two different challenge tests appeared in some lung cancer patients. Also the results of western blot indicated that ATM protein expression in lung cancer patient group was significantly lower than that in control group (i><0.05 ) . Meanwhile, there was a good correlation between DNA repair capacity detected by BLM challenge test and ATM protein expression in lung cancer patients CP<0.01) .Conclusions1. The comet assay or single cell gel eletrophoresis is considered as a simple, rapid and sensitive assay for detecting several types of DNA damage. Combined with mutagen challenge tests, comet assay can be use to measure efficiently individual DNA repair capacity by detecting the dynamic variation of DNA damage.2. The low DNA repair capacity may be an important susceptibility factor for cancers. In this study, it was observed that individuals showed various activities in different DNA repair pathways. Therefore, we suggest that two or more different mutagen challenge tests should be used to detect human DNA repair capacity in molecular epidemiological study. The results obtained from various tests will be more objective than those of single test.3. UVC induces 6-4 photoproducts and cyclobutane pyrimidine dimers (CPD) which are also repaired by NER. BLM is a radiomimetic agent causing mainly double-strand breaks and single-strand breaks are repaired mainly by HRR, NHEJ and BER pathways. ATM protein is at the top of a web of DNA damage responds signaling and is a keyinitiating factor in the cascade of events leading to activation of several DNA damage-responsive signaling pathways. The signal for ATM activation (autophosphorylation) might be chromatin alterations caused by DSBs. Other protein kinases related to ATM carry out similar functions in response to other genotoxic stresses, and some of them collaborate with ATM in the DSB response. In this study, the decreasing ATM protein expression was found may be a risk factor for breast cancer and lung cancer. There was a particular correlation between ATM protein expression level and DRPs measured by BLM challenge test. The present human investigation demonstrated that ATM protein plays a central role in signaling DSBs.