线粒体DNA突变与乳腺癌相关性的研究进展

乳腺癌体细胞线粒体DNA（mtDNA）编码区和调控区会发生多种突变,大量研究提示mtDNA突变积累可能是引起线粒体功能持久缺陷的关键因子,进而导致乳腺癌的发生与进展。本综述归纳了乳腺癌mtDNA点突变、缺失及其拷贝数改变在乳腺癌发生发展、病理生理过程中的作用,讨论了“线粒体——细胞核逆行响应”信号通路及mtDNA作为新的肿瘤标志物和治疗靶标的潜在临床价值。     

线粒体DNA突变增加前列腺癌的发生

越来越多的证据表明，线粒体基因突变与各种癌症有关，已在结肠癌、前列腺癌和其他实体肿瘤中发现各种线粒体DNA编码区和调控区突变。     

线粒体DNA突变与线粒体疾病

线粒体在新陈代谢和生物能量转换中处于中心地位.人体内线粒体DNA(mtDNA)的缺失或突变可导致氧化磷酸化及能量供应异常,从而引起衰老或多种疾病的发生,如肌病、神经疾病等;甚至阿尔茨海默病、帕金森病、亨廷顿舞蹈病及糖尿病等都与线粒体缺陷有关.体内活性氧(ROS)的过量产生是引发线粒体疾病的一个重要因素.     

肺癌线粒体DNA突变的研究

背景与目的：近年来已经在多种肿瘤中发现了线粒体DNA（mitochondrial DNA,mtDNA)突变,但其意义尚不明确。本研究通过检测肺癌组织中的mtDNA突变,探讨mtDNA突变在人肺癌发生中的作用。方法：提取58例肺癌组织及其相应病灶的远端非癌肺组织和外周血淋巴细胞的总DNA（包括核DNA和mtDNA),用巢式PCR（nested PCR)方法扩增相互重叠且涵盖mtDNA全长的58个DNA片段。经PCR产物直接测序法测定mtDNA序列,通过与外周血淋巴细胞比较,确定肺癌组织mtDNA突变。结果：在36例（62．1％）肺癌组织中发现66个突变,其中点突变58个,插入突变4个,缺失突变4个。这些突变分散地分布于mtDNA,以D－loop区突变最多,为18个,在多肽编码区没有明显的热点突变区。在多肽编码区检出的43个点突变中,20个突变不改变氨基酸编码序列。在8例病灶远端非癌肺组织中发现了与相应肿瘤相同的突变。结论：肺癌组织中mtDNA突变可能是随机发生的,且大部分突变对肿瘤的发生可能没有影响,但其有望成为一种肿瘤诊断的分子标记。     

鼻咽癌患者组织及血液中线粒体DNA D-loop区突变

目的:检测鼻咽癌患者线粒体DNA复制控制区(mtDNAD-loop)的突变情况。方法:以健康人血标本和鼻咽癌患者癌旁组织作为对照,对20例鼻咽癌组织样本及相应的血液样本的mtDNAD-loop区进行PCR扩增和直接测序分析。结果:在20份鼻咽癌组织样本中共发现312个核苷酸改变,其中293个为已知的多态性改变,4个为新的未记载过的多态性改变;9个肿瘤组织样本和相应的血标本中共发现15个突变;癌旁组织较多多态性变化,未检出突变;鼻咽癌的mtDNAD-loop区突变率为45%(9/20)。结论:血液中mtDNAD-loop的变异可能与鼻咽癌的易感性有一定的联系;本研究为寻找新的肿瘤基因诊断和肿瘤遗传易感性的标志物提供了依据。     

线粒体DNA突变与实体瘤相关性研究进展

线粒体具有可自主复制的DNA基因组，是细胞内惟一的核外遗传物质。由于其自身特殊的结构特点，线粒体DNA(mtDNA)的突变率比细胞核DNA(nDNA)高10倍以上，而且与细胞凋亡和肿瘤形成都有密切关系，近年来有关各类因素所致mtDNA损伤与细胞癌变、肿瘤发生的相关性研究已成为热点，特别是针对实体瘤的报道逐渐增多，现将有关线粒体基因组的突变、表达异常及不稳定性与实体瘤发生的相关性研究作一综述。     

鼻咽癌组织线粒体DNA突变的研究

[目的]通过检测鼻咽癌组织中线粒体DNA(mitochodrial DNA,mtDNA)部分区域的突变,探讨mtDNA突变在人鼻咽癌发生中的作用.[方法]提取23例鼻咽癌组织及其同一患者外周血白细胞的总DNA,经PCR扩增mtDNA,产物直接测序测定mtDNA序列.[结果]23例鼻咽癌组织中8例(34.8%)发现33个突变,包括点突变27个,插入突变3个,缺失突变3个;这些突变分布于mtDNA序列中,27个mtDNA突变位于D-loop区,其中21个是属于基因库中的多态变化,6个为新发现的突变;另外6个mtDNA突变位于线粒体编码区.[结论]鼻咽癌组织线粒体DNA的D-loop区是一个高度多态性和突变性的区域,mtDNA的突变可能与鼻咽癌的发生有一定的联系,有望成为肿瘤诊断的分子标记.     

线粒体DNA突变与胃癌的关系

线粒体DNA(mtDNA)比核DNA更易受到氧损伤,有较高的突变率.mtDNA改变参与了肿瘤的发生发展,但这些改变在胃癌进展中的作用仍不清楚.胃癌中位点突变和mtDNA含量变化是两种最常见的导致线粒体功能障碍的mtDNA改变.研究mtDNA这两种改变及与胃癌临床病理学特征之间的关系、探讨mtDNA改变在胃癌中的机制是近年来胃癌研究的新方向.     

线粒体DNA突变与实体瘤相关性研究进展

线粒体具有可自主复制的DNA基因组,是细胞内惟一的核外遗传物质.由于其自身特殊的结构特点,线粒体DNA(mtDNA)的突变率比细胞核DNA(nDNA)高1O倍以上,而且与细胞凋亡和肿瘤形成都有密切关系.近年来有关各类因素所致mtDNA损伤与细胞癌变、肿瘤发生的相关性研究已成为热点,特别是针对实体瘤的报道逐渐增多.现将有关线粒体基因组的突变、表达异常及不稳定性与实体瘤发生的相关性研究作一综述.     

线粒体DNA中D-loop区突变在非小细胞肺癌中的意义

目的： 探讨线粒体DNA中D-loop区突变在非小细胞肺癌中的意义及其临床应用的可能性。方法：提取20例非小细胞肺癌患者的癌组织及癌旁组织细胞的线粒体DNA (mtDNA),PCR扩增D-loop区并进行测序,然后以人类基因组中的D-loop区序列为对照,分析基因突变。结果：20例非小细胞肺癌患者的癌组织及癌旁组织mtDNA的D-loop区均发生突变,总突变数265个,其中癌组织特异性突变69个,癌旁组织特异性突变96个,共同突变50个,突变主要集中于D-loop的HVRII区。结论：线粒体DNA中D-loop区的突变主要集中于HVRII区,在非小细胞肺癌的发生发展中可能有重要作用。     

Mitochondrial D310 mutations in the early development of breast cancer

Background:

The role of mitochondrial DNA (mtDNA) mutations in the development of breast cancer is largely unknown. In this study, we investigated the frequency and pattern of mutations in the D310 region, the most commonly mutated region in mtDNA, in a series of breast lesions.

Methods:

Using capillary electrophoresis, we genotyped the D310 sequence of neoplastic epithelial cells from 23 patients with synchronous ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC), 26 patients with IDC only and 29 patients with DCIS only.

Results:

A majority of DCIS (68.4%) and IDC (71.4%) lesions harbour different D310 sequences compared with their matched normal control. Specific D310 sequences were more frequently identified in tumour samples (77.1% of DCIS and 75.5% of IDC) compared with normal tissues (35.3% of normal; P<0.0001). No difference was identified between DCIS lesions with synchronous IDC and those from pure DCIS cases. In five cases, histologically normal tissue adjacent to tumour was found to share D310 sequences with the tumour, while normal tissue taken further away did not.

Conclusion:

Although D310 alterations do not seem to be related to DCIS progression, they were found in histologically normal cells adjacent to tumour. This suggests a field of genetically altered cells, thus D310 mutations could represent a potential marker for the clonal expansion of premalignant breast cancer cells.     

Quantitative analysis of mitochondrial DNA 4977-bp deletion in sporadic breast cancer and benign breast diseases

The mitochondrial DNA (mtDNA) 4977-bp deletion (ΔmtDNA⁴⁹⁷⁷ mutation) is one of the most frequently observed mtDNA mutations in human tissues and may play a role in carcinogenesis. Only a few studies have evaluated ΔmtDNA⁴⁹⁷⁷ mutation in breast cancer tissue, and the findings have been inconsistent, which may be due to methodological differences. In this study, we developed a quantitative real-time PCR assay to assess the level of the ΔmtDNA⁴⁹⁷⁷ mutation in tumor tissue samples from 55 primary breast cancer patients and 21 patients with benign breast disease (BBD). The ΔmtDNA⁴⁹⁷⁷ mutation was detected in all of the samples with levels varying from 0.000149% to 7.0%. The ΔmtDNA⁴⁹⁷⁷ mutation levels were lower in tumor tissues than in adjacent normal tissues in both breast cancer and BBD subjects. The differences, however, were not statistically significant. No significant difference between breast cancer and BBD patients was found in the ΔmtDNA⁴⁹⁷⁷ mutation levels of tumor tissues and adjacent normal tissues. The ΔmtDNA⁴⁹⁷⁷ mutation levels were not significantly associated with clinicopathological characteristics (age, histology, tumor stage, and ER/PR status) in breast cancer or BBD patients. These results do not support the notion that the mitochondrial DNA 4977-bp deletion plays a major role in breast carcinogenesis.     

High frequency of mitochondrial DNA mutations in glioblastoma multiforme identified by direct sequence comparison to blood samples

In an earlier study, we showed that heteroplasmy in the mitochondrial genome of gliomas sometimes occurs in a D-loop polycytosine tract. We extended this study by pairwise comparisons between glioma samples and adjacent brain tissue of 55 patients (50 glioblastomas, 1 astrocytoma WHO grade III, 4 astrocytomas WHO grade II). We used a combination of laser microdissection and PCR to detect and quantify variations in the polycytosine tract. New length variants undetectable in the adjacent brain tissue were observed in 5 glioblastomas (9%). In 2 of these cases, samples from a lower tumor stage (WHO grade II) could be analyzed and revealed the early occurrence of these mutations in both cases. Since the mitochondrial D-loop contains additional repeats and highly polymorphic non-coding sequences, we compared 17 glioblastomas with the corresponding blood samples of the same patients by direct sequencing of the complete D-loop. In 6 of these tumors (35%), instability was detected in 1 or 2 of 3 repeat regions; in 1 of these repeats, the instability was linked to a germline T-to-C transition. Furthermore, of 2 tumors (12%) 1 carried 1 and the other 9 additional transitions. In the latter patient, 6.7 kb of the protein coding mtDNA sequence were analyzed. Six silent transitions and 2 missense mutations (transitions) were found. All base substitutions appeared to be homoplasmic upon sequencing, and 89% occurred at known polymorphic sites in humans. Our data suggest that the same mechanisms that generate inherited mtDNA polymorphisms are strongly enhanced in gliomas and produce somatic mutations.     

胸部放疗致放射性肺炎的相关因素分析

放射性肺炎是胸部放疗的主要剂量限制性因素。严重的RP影响疾病治疗率及预后,目前无有效的治疗或预防措施。早期预测并降低症状性RP的发生对提高胸部放疗疗效意义重大。本篇综述介绍了胸部放疗致RP的有关危险因素与干预措施的相关研究进展。     

食管鳞状上皮细胞癌中P53基因突变的PCR-SSCP检测

应用PCR－SSCP银染技术检测了40例食管鳞状细胞癌p53基因第5、6、7和第8外显于的突变情况。结果显示：13例（32．5％）有p53基因突变，突变主要发生在第5和第8外显子，与癌分化程度无明显关系，但伴淋巴结癌转移病例中p53基因的突变率明显高于无转移者（P＜0．05）。本研究提示，p53基因突变存在于中、晚期食管癌中，并可能与食管鳞癌的进展与转移有关。     

PCR—SSCP法检测非小细胞肺癌p53基因的突变

目的 分析原发性非小细胞肺癌（ＮＳＣＬＣ）中ｐ５３基因５～８外显子点突变发生及意义。方法 动用聚合酶链反应－单链构象多态性分析方法（ＰＣＲ－ＳＳＣＰ分析法）进行检测。结果 发现８例肺良性疾病均无点突变发生,４０例ＮＳＣＬＣ中１９例（４７．５％）有点突变发生,点突变发与性别,年龄,组织学类型和组织发级无明显相关性（Ｐ〉０．０５）,但与临床分期和淋巴结累及状态明显相关（Ｐ〈０．０１,Ｐ〈０．０５）。Ｘ     

甲基丙烯酸环氧丙酯致人支气管上皮恶性转化细胞DNA修复基因点突变的研究

背景与目的： 研究甲基丙烯酸环氧丙酯(glycidyl methacrylate,GMA)致人支气管上皮(16HBE)恶性转化细胞DNA修复基因点突变情况。 材料与方法： 采用聚合酶链式反应-限制性片段长度多态性方法(PCR-RFLP)检测GMA致16HBE恶性转化细胞DNA修复基因hMSH2、XRCC1、XPD及XRCC3的重要位点的突变情况,并以DNA测序方法加以验证。 结果： 16HBE细胞hMSH2 IVS12-6(T>C)位点发生了突变,由野生基因型TT型突变为TC基因型,其它位点未检测到突变。DNA测序结果相符。 结论： 错配修复基因hMSH2 IVS12-6(T>C)位点的突变可能为GMA诱导人支气管上皮细胞恶性转化过程中的重要起始分子事件之一。     

端粒酶抑制剂叠氮胸苷对HeLa细胞放射性DNA损伤修复的影响

背景与目的:研究端粒酶抑制剂叠氮胸苷(Azidothymidine,AZT)对人宫颈癌HeLa细胞DNA放射性损伤修复的影响,探讨端粒酶在放射诱导的DNA损伤修复中的作用.材料与方法:实验分为空白组,AZT组(400 μmol/L AZT处理HeLa细胞24 h),放射组(2 Gy 60Co γ射线照射),AZT放疗组(400 μmol/L AZT处理HeLa细胞24 h后,用2 Gy 60Co γ射线照射).照射后于0、5、10、30、60、180及360 min分别收集细胞,用端粒重复序列扩增法(PCR-based telomeric repeat amplification protocol,TRAP)-联合酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)即TRAP-ELISA法检测端粒酶的活性.用单细胞凝胶电泳法检测DNA单链断裂损伤,以彗尾DNA百分含量表示DNA单链断损伤量.结果:HeLa细胞受2 Gy 60Co γ射线照射后10 min,端粒酶活性即开始增加,60 min后增加明显,360 min时达到最高.AZT处理HeLa细胞后,能使端粒酶活性下降约50%,而且能抑制HeLa细胞照射后端粒酶活性的增加(P＜0.05).单细胞凝胶电泳实验表明,2 Gy 60Co γ射线照射HeLa细胞后0～10 min,AZT放疗组与放射组的彗尾DNA百分含量无明显差异(P＞0.05),照后30～360 min AZT放疗组彗尾DNA百分含量均高于放射组(P均＜0.05).结论:AZT能阻抑照射后30～360 min DNA单链断裂的修复,说明端粒酶可能在放射性DNA损伤修复中具有重要作用.     

Intracranial Germ Cell Tumors: Detection of p53 Gene Mutations by Single-strand Conformation Polymorphism Analysis

Using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis, p53 gene mutation was examined in 12 intracranial germ cell tumors (5 yolk sac carcinomas and 7 germinomas), many of which were derived from young patients in the first to the second decade. A total of 10 mutations were detected in 4 of the 12 cases and, in 3 of them, the mutations were multiple or tandem. Among the 10 mutations, 7 were missense, 1 was splicing and 2 were silent. The 7 missense mutations were located at previously proposed hot spot codons or in their vicinity or, when outside the hot spots, at a codon encoding an amino acid conserved in most vertebrates. These findings suggested that all 7 missense mutations may actually give rise to functional alteration of the p53 protein. The splicing mutation was considered to be a germ-line mutation, though its biological effect was equivocal, since the neoplastic tissue contained an additional mutation. The pattern of the mutations was predominancy of G:C-A:T transition with frequent involvement of the CpG site. These mutations were more frequently detected in yolk sac carcinomas (60%; 3/5 cases) than in germinomas (14%; 1/7 cases), suggesting that the contribution of the p53 mutation to carcinogenesis differed with the histological type of the intracranial germ cell tumor.