癌症患者遗传不稳定性的体内外研究及其与四个DNA修复相关蛋白的关系
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摘要
众所周知人体通过环境中空气、水和食物等介质经常要接触一些有害物质，包括各种潜在致癌或致突变物，例如电离辐射、传染因子和微生物毒素等，长期接触这类致癌物就有可能导致癌症的发生，所以癌症的发生在很大程度上受到环境因素的影响。但是，在接触相同环境的不同个体之间癌症发生的情况又不尽相同，这又与个体的遗传因素有关，因此癌症是机体遗传因素和外界环境因素相互作用的结果，系一种环境相关疾病。然而，在环境因素的作用下，机体的遗传因素会发生改变，比如遗传不稳定性的增加，而癌症发生的一个非常重要的原因就是遗传信息发生较大改变或者是遗传不稳定性增加。因此，对机体遗传不稳定性的研究在环境相关疾病—癌症的研究中就显得非常重要。遗传不稳定性是指可引起一系列能导致总遗传物质发生改变的突变事件的发生率增加的一种短暂或持久的状态，它包括基因水平的核苷酸改变和染色体水平的基因组不稳定性。遗传不稳定性可自发产生，也可由外来物质(比如辐射)暴露所致。电离辐射引起的遗传不稳定性不仅可以影响正常细胞和肿瘤细胞对辐射的反应，也可以影响癌症的发生。电离辐射引起的遗传不稳定性也反映了机体对辐射的敏感性。机体辐射敏感性的研究具有实用意义，如果能在治疗前对癌症患者的辐射敏感性进行预测，这将有助于临床医师制定个体化的治疗计划，有利于提高癌症治疗效率和降低副反应。
维持基因组完整性的主要机制包括两类：(a)DNA复制过程中的高保真机制，这包括保持基因和基因组完整的DNA修复途径和染色体基因外修饰途径；(b)维持有丝分裂的稳定性和染色体的完整性。细胞周期调控在细胞维持基因组完整性中起重要作用，ATM蛋白是一个细胞周期调控蛋白，它可以调节细胞周期、DNA修复和凋亡等过程。当DNA损伤产生后，ATM参与细胞周期调控过程，阻滞细胞分裂以提供足够的时间进行损伤修复。同时ATM激酶会启动一系列的信号转导途径，激活DNA修复和凋亡等过程，从而使损伤得以修复和基因组完整性得以保持。虽然，DNA连接酶只是参与DNA修复的最后步骤—DNA连接，但它们是DNA修复途径以及DNA重组过程中不可缺少的酶，而且在维持基因组完整性中起非常重要的作用。XPF蛋白不仅是核苷酸切除修复途径中切开DNA受损双链过程所需的核酸内切酶之一，而且也是DNA链之间交联的重组修复以及同源重组修复等过程不可缺少的酶。因此，以上这些蛋白都在DNA修复过程起重要作用，在维持机体基因组稳定性中不可缺少。
本论文主要用彗星试验和微核试验检测肺癌和乳腺癌患者遗传损伤的背景值和由辐射引起的遗传损伤，来评价肺癌和乳腺癌患者的遗传不稳定性，并观察这两种癌症患者在遗传稳定性上的个体差异。同时还用western blotting试验检测肺癌和乳腺癌患者ATM、DNA连接酶Ⅲ、DNA连接酶Ⅳ和XPF四个蛋白的表达水平。然后分析肺癌和乳腺癌患者遗传不稳定性与四个DNA修复相关蛋白表达水平之间的关系。本研究分两个阶段完成：
第一阶段：用彗星试验、微核试验和hpry基因突变试验检测放疗期间癌症患者的遗传损伤，其目的是评价这三个试验在检测遗传不稳定性中的灵敏度，并观察癌症患者对放疗反应的个体差异。
第二阶段：肺癌和乳腺癌患者遗传不稳定性和DNA修复相关蛋白表达的研究。
第一部分用彗星试验、微核试验和hprt基因突变试验检测放疗期间癌症患者的遗传损伤
研究自发的以及辐射引起的遗传不稳定性的方法很多，但是克隆存活试验和染色体畸变试验等由于自身的局限性不适合大样本研究。本研究希望通过监测放疗患者的遗传损伤来确定能快速简便地用于检测自发的以及辐射引起的遗传不稳定性的方法。放疗已被广泛用于癌症的治疗，近来在癌症放疗中取得的进展有利于改善癌症患者治疗的预后。但放疗射线在杀伤肿瘤细胞的同时也会对正常细胞造成伤害，从而导致DNA损伤甚至继发肿瘤。对接受放疗后机体产生的生物学效应进行监测可以评价射线对机体产生的损伤。
本研究选择了患有不同癌症的患者24例，对照23例。病例组和对照组在年龄、性别和吸烟等方面均无显著性差异(P＞0.05)。在癌症患者接受放疗前和放疗累积剂量达到10、30和50Gy时采集新鲜外周血，对照组仅采血一次。取1ml外周血分离淋巴细胞用于彗星试验，其余血样分别进行培养用于微核试验和hprt基因突变试验，最后分析癌症患者和对照组遗传损伤背景值是否存在差异，并分析放疗累积剂量和遗传损伤之间的剂量—效应关系。
研究结果显示放疗前癌症患者的平均微核细胞率(MCF)和微核率(MNF)分别为11.29±1.22‰和12.46±1.36‰，而对照组的平均MCF和MNF分别为5.96±0.70‰和6.65±0.82‰，经统计分析，放疗前癌症患者的平均MCF和MNF均明显高于对照组(P＜0.01)。但是，彗星试验的结果显示放疗前癌症患者和对照组的平均尾长(MTL)和平均尾相(MTM)的差异不明显(P＞0.05)，hprt基因突变试验的结果亦显示对照组和病例组之间hprt基因平均突变率(Mf-hprt)无显著性差异(P＞0.05)。癌症患者放疗期间的遗传损伤均随着放疗剂量的增加而增加。在微核试验中，当放疗剂量分别达到10、30和50Gy时平均MCF分别为31.25、53.63和67.28‰，平均MNF分别为35.83、64.63和85.00‰，两者均分别高于0Gy时相应的值(11.29‰和12.46‰，P＜0.01)，而且各剂量组之间也存在显著性差异(P＜0.01)，同时，癌症患者淋巴细胞核分裂指数也随着放疗累积剂量的增加而降低。在彗星试验中，当放疗剂量分别达10、30和50Gy时癌症患者MTL和MTM分别为1.96、2.26和2.63μm以及0.43，0.56和0.70，两者均分别高于0Gy时相应的值(1.77μm和0.70，P＜0.01)，而且各剂量组之间也存在显著性差异(P＜0.01)。我们在hprt基因突变试验中也得到了类似的结果，在放疗剂量为10、30和50Gy时癌症患者的平均Mf-hprt分别为0.91，1.07和1.15‰，均明显(P＜0.01)高于0Gy时的值(0.82‰)，不同剂量组之间也存在显著性差异(P＜0.01)。
本研究结果发现：放疗前癌症患者染色体损伤背景值要高于对照组，并且放疗期间癌症患者的遗传损伤随着放疗剂量的增加而增加，癌症患者对放疗所诱发的遗传损伤存在较大的个体差异。本研究还发现彗星试验、微核试验和hprt基因突变试验均可用于检测放疗引起的遗传损伤，但是hprt基因突变试验的灵敏度不如彗星试验和微核试验高。
第二部分肺癌和乳腺癌患者的遗传不稳定性以及四个DNA修复相关蛋白表达
在第一部分实验中，放疗前癌症患者的遗传损伤在不同试验中结果有差异，其原因可能是样本量比较小以及肿瘤类型较多。因此应该选择单一肿瘤并扩大样本量进行研究。另外，正如其它文献的报道那样，我们发现不同癌症患者对放疗的反应差异很大。因此，如果在放疗前对癌症患者的放射敏感性进行测定，那么，不仅可以提高癌症患者的治疗效率，而且可以同时有效降低放疗的副反应。维持遗传不稳定性的主要机制是机体的DNA修复机制，因此，与DNA修复相关的细胞周期调控蛋白、DNA连接酶和参与多种修复途径的XPF蛋白可能与癌症患者的遗传不稳定性之间存在一定的联系。
为此本研究选择了45例肺癌患者和39例对照(两组人群在年龄、性别、吸烟和饮酒等方面均无显著性差异)，48例乳腺癌患者和相应的对照33例(两组人群在年龄、性别、吸烟和饮酒等方面均无显著性差异)。癌症患者均为新确诊，在未接受治疗之前抽取肝素抗凝静脉血10ml，取4ml血样用于遗传不稳定性研究，其中2ml立即接受3Gy辐射，然后用彗星试验和微核试验分别检测辐射前后的遗传损伤，其余6ml血样用梯度离心法分离淋巴细胞后提取蛋白质，然后用western blotting测定ATM、DNA连接酶Ⅲ、DNA连接酶Ⅳ和XPF四个蛋白的表达水平。
遗传不稳定性检测结果显示，在微核试验中，肺癌患者辐射前的平均MCF和MNF分别为9.84‰和10.72‰，均明显高于对照组相应的值(5.87‰和6.31‰，P＜0.01)，由辐射引起的平均MCF和MNF分别为74.16‰和84.64‰，也同样高于对照组(61.33‰和68.92‰，P＜0.01)。在彗星试验中，肺癌患者辐射前MTL和MTM分别为1.69μm和0.80，明显高于对照组(1.50μm和0.61，P＜0.05)，由辐射引起的MTL和MTM分别为1.46μm和0.96，也分别明显高于对照组(1.05μm和0.65，P＜0.05)。乳腺癌患者遗传不稳定性的检测结果和肺癌患者类似，微核试验和彗星试验的结果均显示乳腺癌患者自发的和由辐射引起的遗传不稳定性均明显高于对照组。我们以对照组辐射引起遗传损伤的百分之九十的百分位数作为分界点将癌症患者分为辐射敏感组和辐射不敏感组。结果显示微核试验对辐射敏感人群的检测具有较高的灵敏度，但是两个试验在检测辐射敏感人群时存在差异，同一患者用不同的试验进行检测可以得到不同的结果。在同一试验中彗星试验的两个指标之间差异较大，而微核试验中的两个指标之间的一致性较高。因此，我们的研究结果提示微核试验可能是较好的用于治疗前癌症患者辐射敏感性测定的方法，但为了较为全面地评价癌症患者的辐射敏感性应该同时使用多种方法。
DNA修复相关蛋白表达结果显示，与对照组相比，癌症患者四个蛋白的表达均有不同程度的下降，但肺癌患者的ATM、DNA连接酶Ⅲ和XPF三个蛋白平均表达水平分别为0.79、0.72和0.72，明显低于对照组(1.18、0.96和0.99)(P＜0.01)，而乳腺癌患者的ATM、DNA连接酶Ⅲ、DNA连接酶Ⅳ和XPF四个蛋白平均表达水平(0.73、0.57、0.71和0.81)均分别明显低于对照组(1.38、0.98、0.95和1.03，P＜0.01)。相关性分析结果显示，彗星试验中的尾长和尾相之间以及微核试验中的微核细胞率和微核率之间均有较好的相关性。尾相测定的结果发现肺癌和乳腺癌患者的遗传损伤背景值与辐射引起的遗传损伤之间均存在较好的相关性。DNA修复相关蛋白表达水平和遗传不稳定性之间的相关性分析发现，在肺癌患者中，ATM蛋白表达水平与尾相测定的遗传损伤背景值以及尾长和尾相测定的辐射引起的遗传损伤之间均有较好的相关性。DNA连接酶Ⅳ蛋白表达水平与尾长测定的遗传损伤背景值以及尾相测定的辐射引起的遗传损伤之间均有一定相关性，XPF蛋白表达水平与尾长和尾相测定的辐射引起的遗传损伤的之间有较好的相关性。在乳腺癌患者中，DNA连接酶Ⅳ蛋白表达水平与尾长测定的遗传损伤背景值以及尾相测定的辐射引起遗传损伤之间均有较好相关性。另外，我们还分析了性别、吸烟和癌症分期等情况对癌症患者遗传不稳定性及DNA修复相关蛋白表达的影响，结果发现吸烟组肺癌患者ATM蛋白表达水平明显低于不吸烟组，Ⅰ期乳腺癌患者DNA连接酶Ⅲ的表达水平均明显高于其余两组，但是Ⅱ、Ⅲ两期乳腺癌患者的DNA连接酶Ⅲ的表达水平无显著性差异。
结论
1．癌症患者在放疗期间所产生的淋巴细胞遗传损伤随着累积放疗剂量的增加而增加，并呈一定的剂量效应关系。癌症患者对放疗的反应存在较大的个体差异。
2．肺癌和乳腺癌患者自发的和由辐射引起的遗传不稳定性均明显高于对照组。肺癌和乳腺癌患者淋巴细胞对辐射的敏感性存在较大的个体差异。
3．彗星试验和微核试验是两种快速、简便、有效的检测癌症患者淋巴细胞自发的以及由辐射引起的遗传不稳定性的方法。这两个方法均可用于癌症患者放疗前淋巴细胞放射敏感性的测定，可为癌症患者治疗方案的制定提供依据。
4．无论肺癌或乳腺癌患者，和对照组相比，四个DNA修复相关蛋白表达均有不同程度的下降，但肺癌患者ATM、DNA连接酶Ⅲ和XPF三个DNA修复相关蛋白表达水平与对照组有明显差异；而乳腺癌患者ATM、DNA连接酶Ⅲ、DNA连接酶Ⅳ和XPF四个DNA修复相关蛋白表达水平与对照组有明显差异。因此，同一蛋白的表达水平在不同的癌症患者中可能有差异。
5．癌症患者DNA修复相关蛋白表达水平的降低可能与其遗传不稳定性的升高之间有一定的相关性，在本研究中也发现了一些相关性，但是由于遗传不稳定性的升高是一个受多方面因素影响的结果，而且与DNA修复相关的蛋白种类很多，所以很可能单个蛋白表达的降低或升高在不同种类的肿瘤或不同的个体有一定的差异。今后的应当同时研究多种细胞周期调控蛋白和与DNA修复相关的蛋白。
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 the
stability 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 assays
There 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 radiotherapy
increased 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 patients
In 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, the
treatment 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 between
these 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 in
phase II or III, but no significant difference was found between breast cancer patients in phase II and breast cancer patients in phaseIII.
Conclusions
1. 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.
维持基因组完整性的主要机制包括两类：(a)DNA复制过程中的高保真机制，这包括保持基因和基因组完整的DNA修复途径和染色体基因外修饰途径；(b)维持有丝分裂的稳定性和染色体的完整性。细胞周期调控在细胞维持基因组完整性中起重要作用，ATM蛋白是一个细胞周期调控蛋白，它可以调节细胞周期、DNA修复和凋亡等过程。当DNA损伤产生后，ATM参与细胞周期调控过程，阻滞细胞分裂以提供足够的时间进行损伤修复。同时ATM激酶会启动一系列的信号转导途径，激活DNA修复和凋亡等过程，从而使损伤得以修复和基因组完整性得以保持。虽然，DNA连接酶只是参与DNA修复的最后步骤—DNA连接，但它们是DNA修复途径以及DNA重组过程中不可缺少的酶，而且在维持基因组完整性中起非常重要的作用。XPF蛋白不仅是核苷酸切除修复途径中切开DNA受损双链过程所需的核酸内切酶之一，而且也是DNA链之间交联的重组修复以及同源重组修复等过程不可缺少的酶。因此，以上这些蛋白都在DNA修复过程起重要作用，在维持机体基因组稳定性中不可缺少。
本论文主要用彗星试验和微核试验检测肺癌和乳腺癌患者遗传损伤的背景值和由辐射引起的遗传损伤，来评价肺癌和乳腺癌患者的遗传不稳定性，并观察这两种癌症患者在遗传稳定性上的个体差异。同时还用western blotting试验检测肺癌和乳腺癌患者ATM、DNA连接酶Ⅲ、DNA连接酶Ⅳ和XPF四个蛋白的表达水平。然后分析肺癌和乳腺癌患者遗传不稳定性与四个DNA修复相关蛋白表达水平之间的关系。本研究分两个阶段完成：
第一阶段：用彗星试验、微核试验和hpry基因突变试验检测放疗期间癌症患者的遗传损伤，其目的是评价这三个试验在检测遗传不稳定性中的灵敏度，并观察癌症患者对放疗反应的个体差异。
第二阶段：肺癌和乳腺癌患者遗传不稳定性和DNA修复相关蛋白表达的研究。
第一部分用彗星试验、微核试验和hprt基因突变试验检测放疗期间癌症患者的遗传损伤
研究自发的以及辐射引起的遗传不稳定性的方法很多，但是克隆存活试验和染色体畸变试验等由于自身的局限性不适合大样本研究。本研究希望通过监测放疗患者的遗传损伤来确定能快速简便地用于检测自发的以及辐射引起的遗传不稳定性的方法。放疗已被广泛用于癌症的治疗，近来在癌症放疗中取得的进展有利于改善癌症患者治疗的预后。但放疗射线在杀伤肿瘤细胞的同时也会对正常细胞造成伤害，从而导致DNA损伤甚至继发肿瘤。对接受放疗后机体产生的生物学效应进行监测可以评价射线对机体产生的损伤。
本研究选择了患有不同癌症的患者24例，对照23例。病例组和对照组在年龄、性别和吸烟等方面均无显著性差异(P＞0.05)。在癌症患者接受放疗前和放疗累积剂量达到10、30和50Gy时采集新鲜外周血，对照组仅采血一次。取1ml外周血分离淋巴细胞用于彗星试验，其余血样分别进行培养用于微核试验和hprt基因突变试验，最后分析癌症患者和对照组遗传损伤背景值是否存在差异，并分析放疗累积剂量和遗传损伤之间的剂量—效应关系。
研究结果显示放疗前癌症患者的平均微核细胞率(MCF)和微核率(MNF)分别为11.29±1.22‰和12.46±1.36‰，而对照组的平均MCF和MNF分别为5.96±0.70‰和6.65±0.82‰，经统计分析，放疗前癌症患者的平均MCF和MNF均明显高于对照组(P＜0.01)。但是，彗星试验的结果显示放疗前癌症患者和对照组的平均尾长(MTL)和平均尾相(MTM)的差异不明显(P＞0.05)，hprt基因突变试验的结果亦显示对照组和病例组之间hprt基因平均突变率(Mf-hprt)无显著性差异(P＞0.05)。癌症患者放疗期间的遗传损伤均随着放疗剂量的增加而增加。在微核试验中，当放疗剂量分别达到10、30和50Gy时平均MCF分别为31.25、53.63和67.28‰，平均MNF分别为35.83、64.63和85.00‰，两者均分别高于0Gy时相应的值(11.29‰和12.46‰，P＜0.01)，而且各剂量组之间也存在显著性差异(P＜0.01)，同时，癌症患者淋巴细胞核分裂指数也随着放疗累积剂量的增加而降低。在彗星试验中，当放疗剂量分别达10、30和50Gy时癌症患者MTL和MTM分别为1.96、2.26和2.63μm以及0.43，0.56和0.70，两者均分别高于0Gy时相应的值(1.77μm和0.70，P＜0.01)，而且各剂量组之间也存在显著性差异(P＜0.01)。我们在hprt基因突变试验中也得到了类似的结果，在放疗剂量为10、30和50Gy时癌症患者的平均Mf-hprt分别为0.91，1.07和1.15‰，均明显(P＜0.01)高于0Gy时的值(0.82‰)，不同剂量组之间也存在显著性差异(P＜0.01)。
本研究结果发现：放疗前癌症患者染色体损伤背景值要高于对照组，并且放疗期间癌症患者的遗传损伤随着放疗剂量的增加而增加，癌症患者对放疗所诱发的遗传损伤存在较大的个体差异。本研究还发现彗星试验、微核试验和hprt基因突变试验均可用于检测放疗引起的遗传损伤，但是hprt基因突变试验的灵敏度不如彗星试验和微核试验高。
第二部分肺癌和乳腺癌患者的遗传不稳定性以及四个DNA修复相关蛋白表达
在第一部分实验中，放疗前癌症患者的遗传损伤在不同试验中结果有差异，其原因可能是样本量比较小以及肿瘤类型较多。因此应该选择单一肿瘤并扩大样本量进行研究。另外，正如其它文献的报道那样，我们发现不同癌症患者对放疗的反应差异很大。因此，如果在放疗前对癌症患者的放射敏感性进行测定，那么，不仅可以提高癌症患者的治疗效率，而且可以同时有效降低放疗的副反应。维持遗传不稳定性的主要机制是机体的DNA修复机制，因此，与DNA修复相关的细胞周期调控蛋白、DNA连接酶和参与多种修复途径的XPF蛋白可能与癌症患者的遗传不稳定性之间存在一定的联系。
为此本研究选择了45例肺癌患者和39例对照(两组人群在年龄、性别、吸烟和饮酒等方面均无显著性差异)，48例乳腺癌患者和相应的对照33例(两组人群在年龄、性别、吸烟和饮酒等方面均无显著性差异)。癌症患者均为新确诊，在未接受治疗之前抽取肝素抗凝静脉血10ml，取4ml血样用于遗传不稳定性研究，其中2ml立即接受3Gy辐射，然后用彗星试验和微核试验分别检测辐射前后的遗传损伤，其余6ml血样用梯度离心法分离淋巴细胞后提取蛋白质，然后用western blotting测定ATM、DNA连接酶Ⅲ、DNA连接酶Ⅳ和XPF四个蛋白的表达水平。
遗传不稳定性检测结果显示，在微核试验中，肺癌患者辐射前的平均MCF和MNF分别为9.84‰和10.72‰，均明显高于对照组相应的值(5.87‰和6.31‰，P＜0.01)，由辐射引起的平均MCF和MNF分别为74.16‰和84.64‰，也同样高于对照组(61.33‰和68.92‰，P＜0.01)。在彗星试验中，肺癌患者辐射前MTL和MTM分别为1.69μm和0.80，明显高于对照组(1.50μm和0.61，P＜0.05)，由辐射引起的MTL和MTM分别为1.46μm和0.96，也分别明显高于对照组(1.05μm和0.65，P＜0.05)。乳腺癌患者遗传不稳定性的检测结果和肺癌患者类似，微核试验和彗星试验的结果均显示乳腺癌患者自发的和由辐射引起的遗传不稳定性均明显高于对照组。我们以对照组辐射引起遗传损伤的百分之九十的百分位数作为分界点将癌症患者分为辐射敏感组和辐射不敏感组。结果显示微核试验对辐射敏感人群的检测具有较高的灵敏度，但是两个试验在检测辐射敏感人群时存在差异，同一患者用不同的试验进行检测可以得到不同的结果。在同一试验中彗星试验的两个指标之间差异较大，而微核试验中的两个指标之间的一致性较高。因此，我们的研究结果提示微核试验可能是较好的用于治疗前癌症患者辐射敏感性测定的方法，但为了较为全面地评价癌症患者的辐射敏感性应该同时使用多种方法。
DNA修复相关蛋白表达结果显示，与对照组相比，癌症患者四个蛋白的表达均有不同程度的下降，但肺癌患者的ATM、DNA连接酶Ⅲ和XPF三个蛋白平均表达水平分别为0.79、0.72和0.72，明显低于对照组(1.18、0.96和0.99)(P＜0.01)，而乳腺癌患者的ATM、DNA连接酶Ⅲ、DNA连接酶Ⅳ和XPF四个蛋白平均表达水平(0.73、0.57、0.71和0.81)均分别明显低于对照组(1.38、0.98、0.95和1.03，P＜0.01)。相关性分析结果显示，彗星试验中的尾长和尾相之间以及微核试验中的微核细胞率和微核率之间均有较好的相关性。尾相测定的结果发现肺癌和乳腺癌患者的遗传损伤背景值与辐射引起的遗传损伤之间均存在较好的相关性。DNA修复相关蛋白表达水平和遗传不稳定性之间的相关性分析发现，在肺癌患者中，ATM蛋白表达水平与尾相测定的遗传损伤背景值以及尾长和尾相测定的辐射引起的遗传损伤之间均有较好的相关性。DNA连接酶Ⅳ蛋白表达水平与尾长测定的遗传损伤背景值以及尾相测定的辐射引起的遗传损伤之间均有一定相关性，XPF蛋白表达水平与尾长和尾相测定的辐射引起的遗传损伤的之间有较好的相关性。在乳腺癌患者中，DNA连接酶Ⅳ蛋白表达水平与尾长测定的遗传损伤背景值以及尾相测定的辐射引起遗传损伤之间均有较好相关性。另外，我们还分析了性别、吸烟和癌症分期等情况对癌症患者遗传不稳定性及DNA修复相关蛋白表达的影响，结果发现吸烟组肺癌患者ATM蛋白表达水平明显低于不吸烟组，Ⅰ期乳腺癌患者DNA连接酶Ⅲ的表达水平均明显高于其余两组，但是Ⅱ、Ⅲ两期乳腺癌患者的DNA连接酶Ⅲ的表达水平无显著性差异。
结论
1．癌症患者在放疗期间所产生的淋巴细胞遗传损伤随着累积放疗剂量的增加而增加，并呈一定的剂量效应关系。癌症患者对放疗的反应存在较大的个体差异。
2．肺癌和乳腺癌患者自发的和由辐射引起的遗传不稳定性均明显高于对照组。肺癌和乳腺癌患者淋巴细胞对辐射的敏感性存在较大的个体差异。
3．彗星试验和微核试验是两种快速、简便、有效的检测癌症患者淋巴细胞自发的以及由辐射引起的遗传不稳定性的方法。这两个方法均可用于癌症患者放疗前淋巴细胞放射敏感性的测定，可为癌症患者治疗方案的制定提供依据。
4．无论肺癌或乳腺癌患者，和对照组相比，四个DNA修复相关蛋白表达均有不同程度的下降，但肺癌患者ATM、DNA连接酶Ⅲ和XPF三个DNA修复相关蛋白表达水平与对照组有明显差异；而乳腺癌患者ATM、DNA连接酶Ⅲ、DNA连接酶Ⅳ和XPF四个DNA修复相关蛋白表达水平与对照组有明显差异。因此，同一蛋白的表达水平在不同的癌症患者中可能有差异。
5．癌症患者DNA修复相关蛋白表达水平的降低可能与其遗传不稳定性的升高之间有一定的相关性，在本研究中也发现了一些相关性，但是由于遗传不稳定性的升高是一个受多方面因素影响的结果，而且与DNA修复相关的蛋白种类很多，所以很可能单个蛋白表达的降低或升高在不同种类的肿瘤或不同的个体有一定的差异。今后的应当同时研究多种细胞周期调控蛋白和与DNA修复相关的蛋白。
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 the
stability 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 assays
There 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 radiotherapy
increased 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 patients
In 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, the
treatment 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 between
these 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 in
phase II or III, but no significant difference was found between breast cancer patients in phase II and breast cancer patients in phaseIII.
Conclusions
1. 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.
引文
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