| It is well known that human bodies are usually exposed to some hazardous substances in air, water and food, which include various kinds of potential carcinogens or mutagens, such as ionizing radiation, infection factors and microorganism toxin. Continuous exposure to these substances may result in the development of cancers, so the development of cancer is influenced greatly by environmental factors. However, the occurrence of cancer is different among individuals who are exposed to the similar environmental condition, which may be due to the genetic factors. Hence, cancer development is the result of the interaction between environmental factors and human inherent factors, and cancer is an environmentally associated disease. But the genetic factors will alter, for example, genetic instability increases, under the action of environmental factors, and one important reason for cancer is that the genetic information changes greatly or the genetic instability increases. So the study of genetic instability is very important to study the environmentally associated disease-cancer. Genetic instability refers to a temporary or permanent state which can induce the increase of occurrence rate of mutation event resulting in the alteration of total genetic substances. Genetic instability includes nucleotide alteration at the gene level and genomic instability at the chromosomal level, and it can be spontaneous or induced by environmental factors, such as ionizing radiation. The genetic instability induced by ionizing radiation can influence not only the response of normal cells and tumor cells to radiation, but also the development of cancer. The genetic instability induced by ionizing radiation also reflects individual radiosensitivity. The study on the cellular radiosensitivity is useful, if the radiosensitivity of cancer patients can be predicted before treatment, it will be helpful for clinicians to establish individualized therapeutic protocol, and it will also improve the treatment efficiency of cancer and reduce side effects.The main mechanisms maintaining genomic integrity include following: (a) DNA repair mechanism, which contains DNA repair pathway maintaining the integrity of gene and genome and chromosomal modification pathway; (b) maintenance of thestability of mitosis and the integrity of chromosome. Cell cycle regulation plays an important role in keeping genomic integrity. ATM protein takes part in cell cycle regulation, which can regulate the processes of cell cycle checkpoints, DNA repair, and cell apoptosis. When the DNA is damaged, ATM protein is activated and the activated ATM protein initiates the cell cycle regulation, blocks the cell division supporting enough time for DNA repairing. ATM kinase can activate series of signal transduction pathways and the processes of DNA repair and apoptosis, and then the damaged DNA is repaired and the integrity of genome is maintained. Although, DNA ligases only take part in the last step of DNA repairing-DNA joining, the enzymes are necessary for DNA repairing and DNA recombination and they also play an important role in maintaining genome integrity. XPF protein is not only one of the indispensable endonucleases splitting double strands of damage DNA in the nucleotide excision repair (NER) pathway, but also the necessary enzyme for repairing cross-link of DNA strands by recombination repair and homozygous recombination (HR) repair pathways. Therefore, these proteins play an important role in DNA repairing, and they are necessary for maintaining genomic integrity.The background and ionizing radiation induced genetic damage of lung and breast cancer patients was detected using comet and micronucleus (MN) assays in this study, and the genetic instability of lung and breast cancer patients was assessed, also the inter-individual variation of genetic instability among cancer patients was observed. In addition, the expression levels of ATM, DNA ligaseIII, DNA ligaseIV, and XPF proteins of lung and breast cancer patients were detected with western blotting. Then, the relationship between genetic instability and expression levels of four DNA repair related proteins of lung and breast cancer patients was analyzed. The study consisted of two parts:Part I : The genetic damage of cancer patients during radiotherapy was measured with comet, micronucleus, and hprt gene mutation assays. The aim of this part was to assess the sensitivity of these three assays in detecting genetic instability, and to observe the inter-individual variation of response to radiotherapy in cancer patients.Part II: The genetic instability and expression levels of four DNA repair related proteins of lung and breast cancer patients were investigated.Part I The genetic damage of cancer patients during radiotherapy was measured with comet, micronucleus, and hprt gene mutation assaysThere are several methods for studying spontaneous and ionizing radiation induced genetic instability. The clone survival and chromosomal aberration assays was not suitable for large scale investigation due to some limitations. The aim of this part of our study was to establish rapid and simple methods for detecting spontaneous and ionizing radiation induced genetic instability through monitoring genetic damage of cancer patients during radiotherapy. Radiotherapy was widely used in the treatment of cancer, and the improvement of radiotherapy will be propitious to the prognosis of cancer. But the radiation used in radiotherapy will injure the normal cells when it kill the tumor cells, and then result in DNA damage and even occurrence of second tumor. The damage induced by radiation in individuals can be assessed by monitoring the biological effect after radiotherapy.Twenty four patients with various cancers and 23 controls were selected in this study, and there was no significant difference of age, sex, and smoking habit between patients and controls (P>0.05). Fresh peripheral blood was collected from cancer patients when patients received the cumulative doses of radiotherapy at 0, 10, 30, and 50Gy. The blood from controls was collected only one time. Lymphocytes were separated from 1ml of blood sample, and it was used in comet assay, and the rest blood was used for cell culture of micronucleus (MN) and hprt gene mutation assays. The difference of background genetic damage was analyzed between patients and controls, and the dose-effect relationship between cumulative doses of radiotherapy and genetic damage was assessed.The results indicated that the micronucleated cell frequency (MCF) and micronuclei frequency (MNF) of cancer patients before radiotherapy were 11.29±1.22 ‰ and12.46±1.36‰, and it were 5.96±0.70‰ and 6.65±0.82‰ in controls, and statistic analysis indicated that the average MCF and MNF of cancer patients before radiotherapy were significantly higher than those of controls (P<0.01). However, the results of comet assay showed that the differences of mean tail length (MTL) and mean tail moment (MTM) between cancer patients and controls were not significant (P>0.05), and the results of hprt gene mutation assay also indicated that there was no significant difference of mutant frequency of hprt gene between cancer patients and controls (P>0.05). The genetic damage of cancer patients during radiotherapyincreased with the cumulative doses of radiotherapy. In the MN assay, the mean values were 31.25, 53.63, and 67.28 ‰ for MCF, and 35.83, 64.63, and 85.00 ‰ for MNF, respectively, when the cumulative doses were 10, 30, and 50 Gy, respectively, which was significantly higher than the corresponding values of 0 Gy (11.29‰ and 12.46‰, P<0.01) , and there was significant difference between various dose groups (P<0.01), at the same time, the nucleus division index (NDI) of lymphocytes in cancer patients decreased with the increase of cumulative doses. In the comet assay, the mean values were 1.96, 2.26, and 2.63μm for MTL, and 0.43, 0.56, and 0.70 for MTM, respectively, when the cumulative doses were 10, 30, and 50 Gy, respectively, which were significantly higher than the corresponding values of 0 Gy (1.77μm and 0.70, P<0.01), and there was also significant difference between various dose groups (P<0.01). Similar results were obtained in the hprt gene mutation assay, the mean Mfs-hprt were 0.91, 1.07 and 1.15 ‰, respectively, when the cumulative doses were 10, 30, and 50 Gy, respectively, which were significantly higher than the value of 0 Gy (0.82‰, P<.01), and there was also significant difference between various dose groups (P<.01).The results of our study suggested that the background genetic damage of cancer patients before radiotherapy was higher than that of controls, and the genetic damage of cancer patients during radiotherapy increased with cumulative doses of radiotherapy, also there was wide variation of genetic damage induced by radiotherapy among cancer patients. In addition, comet, MN, and hprt gene mutation assays can be used to detect genetic damage induced by radiotherapy, but the hprt gene mutation assay was not as sensitive as comet and MN assays.Part II The genetic instability and the expression levels of four DNA repair related proteins were studied in lung and breast cancer patientsIn the first part of our study, the results of genetic damage of cancer patients during radiotherapy showed slight difference among three assays, which may be due to the small sample size and various types of cancer. Therefore, the patients with single tumor should be selected and the sample size should be enlarged in next investigation. In addition, just like the results reported by other literatures, it was also found that there was wide variation of response to radiotherapy in cancer patients. If the radiosensitivity of cancer patients can be measured before radiotherapy, thetreatment of cancer will be more efficient and the side effects of radiotherapy will be reduced effectively. DNA repair was the major mechanism maintaining genetic integrity, hence, those proteins related to DNA repair, such as cell cycle regulatory proteins and DNA ligases, and XPF protein taking part in several DNA repair pathways may have some links with genetic instability.In the second part of the present study, 45 lung cancer patients and 39 controls, and 48 breast cancer patients and 33 controls were selected, and there was no significant difference of age, sex, smoking habit, and alcohol consumption between cancer patients and controls. All cancer patients were untreated in our study, 10ml of heparinized venous blood was collected from cancer patients before treatment, each blood sample was divided in to three parts, one part (2ml of blood) was not exposed to radiation (non-irradiated sample), the second part (2ml of blood) was exposed to 3Gy of radiation (irradiated sample), The third part (6ml of blood) was used for protein extraction. The genetic damage of non-irradiated and irradiated samples was measured with comet and MN assays. The expression levels of ATM, DNA ligaseIII, DNA ligaseIV, and XPF proteins were detected with western blotting.The results of genetic instability showed that in the MN assay, the mean values of MCF and MNF of non-irradiated samples in lung cancer patients were 9.84‰ and 10.72‰, respectively, which were significantly higher than those of controls (5.87‰ and 6.3 l‰, P < 0.01), and the mean values of MCF and MNF of irradiated samples in lung cancer patients were 74.16‰ and 84.64‰, respectively, which were also significantly higher than those of controls (61.33‰ and 68.92‰, P < 0.01); in the comet assay the MTL and MTM of non-irradiated samples in lung cancer patients were 1.69μm and 0.80, which were significantly higher than those of controls (1.50μm and 0.61, P < 0.05), and the MTL and MTM of irradiated samples in lung cancer patients were 1.46μm and 0.96, which were also significantly higher than those of controls (1.05μm and 0.65, P < 0.05). The results of genetic instability in breast cancer patients were similar to that in lung cancer patients. The results of both comet assay and MN assay suggested that the spontaneous and irradiation induced genetic instability of breast cancer patients was significantly higher than that of controls. Patients were divided into two groups, i.e. sensitive group and non-sensitive group, by the 90 percentiles of IR-induced parameters. The results indicated that MN assay is more sensitive in assessing radiosensitive population, and there is different betweenthese two assays in assessing radiosensitive population. More differenece was found between two indexes used in the comet assay. And the indexes used in MN assay were more consistent. Hence, we suggested that the MN assay was a better method for detecting radiosensitivity of cancer patients before treatment. However, more methods should be simultaneously used to assess the radiosensitivity of cancer patients.The results of the expression levels of proteins related to DNA repair indicated that the expression levels of four proteins in cancer patients decreased to different extent as compared with controls. The expression levels of ATM, DNA ligaselll, and XPF of lung cancer patients were 0.79, 0.72 and 0.72, respectively, which was significantly lower than those of controls (1.18, 0.96, and 0.99, P < 0.01).The expression levels of ATM, DNA ligaseIII, DNA ligaseIV, and XPF four proteins of breast cancer patients (0.73 , 0.57, 0.71, and 0.81) were significantly lower than those of controls (1.38,098, 0.95, and 1.03, P < 0.01). The results of correlation analysis showed that good correlations were found between MTL and MTM in the comet assay and between MCF and MNF in the MN assay. The results of MTM indicated that there were good correlations between background genetic damage and irradiation induced genetic damage wherever in lung cancer patients and in breast cancer patients. The correlation analysis between genetic instability and expression levels of DNA repair related proteins indicated that in lung cancer patients there were good correlations between expression level of ATM protein and background and irradiation induced genetic damage measured with MTM, and between expression level of DNA ligaseIV and background genetic damage measured with MTL and irradiation induced genetic damage measured with MTM, and between expression level of XPF protein and irradiation induced genetic damage by MTL and MTM; in breast cancer patients the good correlations were found between expression level of DNA ligaselV and background genetic damage measured with MTL and irradiation induced genetic damage measured with MTM. In addition, the influence of sex, smoking habit, and cancer classification on genetic instability and expression level of DNA repair related proteins of cancer patients was analyzed, and the results indicated that the expression level of ATM protein in smokers of lung cancer patients was significantly lower than that in non-smokers of lung cancer patients, and the expression level of DNA ligaselll of breast cancer patients in phase I was significantly higher than those patients inphase II or III, but no significant difference was found between breast cancer patients in phase II and breast cancer patients in phaseIII.Conclusions1. The genetic damage of lymphocytes from cancer patients during radiotherapy increased with cumulative doses of radiation, and there is a dose-effect relationship between genetic damage and cumulative doses of radiation. There was wide variation of response to radiotherapy in cancer patients.2. The spontaneous and irradiation induced genetic instability of lung and breast cancer patients is significantly higher than that of controls. Wide inter-individual difference of cellular response to ionizing radiation is found in lymphocytes from lung and breast cancer patients.3. Both comet assay and MN assay are rapid, simple, and efficient methods in detecting spontaneous and irradiation induced genetic instability of lymphocytes from cancer patients. These two methods can be used to detect radiosensitivity of lymphocytes from cancer patients before treatment, which may provide evidence for establishing treatment protocols for cancer patients.4. The expression levels of four DNA repair related proteins decrease to some extent, whatever in lung or breast cancer patients as compared with controls. However, the expression levels of ATM, DNA ligaseIII, and XPF three proteins of lung cancer patients are significantly lower than those of controls. While the expression level of ATM, DNA ligaseIII, DNA ligaseIV,and XPF four proteins of breast cancer patients are significantly lower than those of controls. Therefore, the expression level of the same protein may be different in patients with different types of cancers.5. Decrease of expression level of DNA repair related proteins in cancer patients may have correlations with increase of genetic instability, and some correlations are found in the present study, but the genetic instability is influenced by multiple factors. Moreover, there are many proteins which are related to DNA repair. Therefore, the expression level of single protein may be different in different individuals or in different types of cancers. We suggest that more cell cycle regulatory proteins and DNA repair related proteins should be investigated in future research. |
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