结晶型NiS诱发恶性转化细胞中的p16基因和FHIT基因的变化

目的 检测结晶型NiS诱发永生化人支气管上皮细胞系(16HBE)恶性转化过程中脆性组氨酸三联体基因(FHIT)和p16基因的变化,探讨镍化合物致癌的分子机制。方法 采用RT-PCR、DNA序列分析、银染PCR—SSCP方法检测恶性转化细胞、成瘤细胞中FHIT基因和p16基因的变化。结果 与对照组16HBE相比,转化细胞、成瘤细胞p16基因第2外显子、第2-3外显子末见突变,mRNA表达末见异常;FHIT基因第5,6,7,8外显子及第1-4外显子、第5-9外显子末见突变,但发现转化细胞、成瘤细胞FHIT基因的mRNA失表达或出现异常转录本;对其中FHIT基因第5-9外显子的一异常转录本测序分析显示,FHIT第6,7,8外显子缺失,第5,9外显子异常拼接,且中间插入一个36bp的小片段。结论 在结晶型NiS诱发16HBE恶性转化过程中,p16基因在DNA和mRNA水平末见异常改变,提示p16基因在镍致癌过程中可能不发挥作用;FHIT基因在mRNA水平出现缺失或异常转录本,表明FHIT基因在镍致癌过程中可能发挥一定作用;镍诱导的FHIT基因异常,可能是将外源致癌物、染色体脆性部位不稳定性和细胞恶变相联系起来的一个分子事件,FHIT基因可能是镍等外源致癌物作用的主要靶基因之一。     

结晶型NiS诱发人支气管上皮细胞系恶性转化

目的:研究结晶型硫化镍(NiS)诱发SV-40 Large T抗原永生化的人支气管上皮细胞系(16HBE)恶性转化作用.方法:体外用不同浓度结晶型NiS多次处理16HBE细胞,软琼脂集落形成和裸鼠成瘤试验鉴定转化细胞的恶性度.结果:细胞培养至35代,发现NiS可诱导16HBE恶性转化.转化细胞排列紊乱,重叠生长,在软琼脂中的集落形成率显著高于对照组(P＜0.01),并呈时间剂量依赖关系;转化细胞在裸鼠体内成瘤,组织学证实为低分化鳞状细胞癌.结论:结晶型NiS有较强的诱导人支气管上皮细胞恶性转化能力；该转化模型为镍致癌机制的分子水平研究提供了理想的研究系统。     

甲基丙烯酸环氧丙酯致人支气管上皮细胞恶性转化过程中DNMT1及MBD1基因表达的变化

目的：分析甲基丙烯酸环氧丙酯（glycidyl methacrylate,GMA）致人支气管上皮16HBE细胞恶性转化过程中不同时点甲基化转移酶及甲基CpG结合蛋白家族基因的表达差异,初步探讨GMA诱导16HBE细胞发生恶性转化的表观遗传机制。方法：采用人类全基因组表达谱芯片筛选GMA诱导16HBE细胞恶性转化过程中甲基化转移酶及甲基CpG结合蛋白家族的差异表达基因,并采用实时定量PCR（Real time PCR）技术检测筛选所得差异基因DNMT1及MBD1 mRNA的表达情况。结果：芯片结果显示在转化第10代（前期）细胞中DNMT1及MBD1基因较同代龄对照细胞表达上调,实时定量PCR进一步确证DNMT1及MBD1基因表达分别上调80.60%、79.10%,与芯片结果一致。结论：甲基化可能是GMA致16HBE细胞发生恶性转化的一种机制,DNMT1及MBD1基因可能是多个甲基化相关基因转录表达下调的关键调控点,其在GMA致16HBE细胞恶性转化过程中起着重要作用。     

硫化镍转化人支气管上皮细胞基因差异表达芯片的研究

背景与目的近年研究发现硫化镍(NiS)可引起人支气管上皮细胞(humanbronchialepithelialcell,16HBE)发生恶性转化及转化细胞致癌性,其机制可能与NiS引起基因突变、多种转录因子的异常表达有关。为此,本研究利用NiS恶性转化的体外培养细胞模型,用cDNA微阵列技术研究经NiS转化后的16HBE细胞基因的差异表达,从基因组水平探讨NiS所致细胞恶变的相关基因差异改变。方法分别提取16HBE和经NiS单独处理发生转化的NiS16HBE细胞的总RNA,逆转录合成cDNA并以Cy3dCTP和Cy5dCTP荧光素分别标记制作探针。探针混合后与含4000个人类基因的芯片杂交。以ScanArray4000扫描仪扫描芯片,以GenPixPro3.0软件分析荧光信号,然后对差异表达的基因进行生物学信息分析。结果NiS16HBE细胞样本与16HBE细胞样本比较,呈现差异表达的基因有151个(3.78%),其中81个(53.64%)上调,70个(46.36%)下调。结论NiS的转化作用与应激反应基因、免疫相关基因、DNA合成和修复基因、代谢相关基因、原癌基因和抑癌基因等多类基因的异常表达有关。     

叶绿酸抗细胞恶变相关cDNA序列的克隆

目的:克隆叶绿酸抑制人支气管上皮细胞系(16HBE)恶性转化的相关差异表达cDNA序列。方法:以叶绿酸及反式-BPDE共同处理的16HBE细胞为实验细胞,以反式-BPDE单独处理的16HBE细胞为对照细胞,采用cDNA代表性差异分析法,比较2种细胞基因表达的差异,分离叶绿酸抗反式-BP-DE作用的相关差异表达cDNA片段。分离的片段分别与pGEM-T载体连接并转化入JM109细菌中,对质粒DNA进行测序。以BLASTN程序进行GenBank的同源性检索。结果:克隆的5条cDNA序列中,有3条为新基因序列,另2条分别与类S18/S6人核糖体蛋白及alpha-烯醇酶等有同源性。结论:克隆的5条cDNA序列所在的基因可能与叶绿酸抗细胞恶性转化分子机制密切相关。     

氯化镉诱发16HBE细胞恶性转化中hOGG1基因表达和序列变化的研究

目的：探讨氯化镉诱发人支气管上皮（16HBE）细胞系恶性转化过程中hOGG1基因mRNA和蛋白动态变化的规律及其序列变化情况。方法：用反转录-聚合酶链反应（PT-PCR）和免疫组织化学（SP法）检测16HBE细胞、氯化镉恶性转化16HBE细胞系不同阶段（第5、15和35代细胞及成瘤细胞）hOGG1基因mRNA和蛋白的表达情况；用直接测序法分析hOGG1基因第7外显子的序列情况。结果：随着转化代数的增加，h0GGl基因mRNA和蛋白的表达逐渐降低，到第15代细胞后表达明显下降（P〈0．05）；hOGG1基因第7外显子第162位点插入一个腺嘌呤A，为移码突变。结论：hOGG1基因水平的下调和突变可能是氯化镉分子致癌的重要机制。     

胃癌环氧化酶-2 、hMLH1 及hMSH2 微卫星不稳定与基因调控的关系

 目的 检测临床手术切除43例胃癌及相应正常组织COX-2、hMLH1和hMSH2微卫星不稳定状态MSI及三种基因启动子甲基化情况，并进一步探讨它们与胃癌发生的关系。方法 采用聚合酶链反应（PCR）技术检测5个位点的MSI状态；甲基化特异性PCR（Methylation specific PCR，MSP）方法检测胃癌及正常组织COX-2、hMLH1及hMSH2三种基因启动子CpG岛甲基化状态。结果 43例胃癌中MSI总检出率为48．84％（21／43），五个位点的MSI检出率无显著差别。COX-2和hMLH1基因启动子CpG岛甲基化在43例胃癌中分别有8例和13例，正常组织中未检测到。hMSH2基因启动子CpG岛在胃癌及正常组织中均未检测到甲基化。在MSI-H组hMLH1基因启动子CpG岛甲基化率显著高于MSS组（P〈0．01）；而在MSI-H和MSI-L组间以及MSI-L和MSS无差别。MSI-H组中COX-2基因启动子CpG岛甲基化8例，且有7例胃癌同时出现hMLH1和COX-2基因启动子CpG岛甲基化。结论 在MSI胃癌（尤其是MSI-H型胃癌）的发生、发展过程中可能同时出现了hMLH1和COX-2基因启动子CpG岛的甲基化（即表型遗传修饰）。MSI胃癌hMLH1基因启动子CpG岛甲基化率高于MSS胃癌，提示检测hMLH1基因启动子CpG岛甲基化对于判断肿瘤类型有一定意义。     

自噬途径对反式-BPDE诱导人支气管上皮细胞16HBE影响及其机制的探讨

目的:研究反式-BPDE诱导正常的人支气管上皮细胞16HBE发生恶性转化前后自噬水平的变化及其可能机制。方法:利用前期建立的细胞恶性转化模型,采用激光共聚焦扫描显微镜成像技术,在无损伤状态下,实时观测反式-BPDE恶性转化细胞(16HBE-T)和正常细胞(16HBE-N)中自噬体标记蛋白GFP-LC3的荧光强度及定位,并利用蛋白质印迹法技术检测细胞内自噬信号通路关键效应分子的表达。结果:16HBE-T细胞与16HBE-N细胞相比,GFP-LC3蛋白的点状聚集明显减少,只有16HBE-N细胞的1/3左右,表明自噬水平下降,蛋白质印迹法检测发现,mTOR激酶活性上升,Beclin 1表达下降。结论:反式-BPDE可能通过自噬途径参与诱导16HBE细胞恶性转化的癌变过程,将为预防和治疗肺癌提供重要信息。     

氯化镉恶性转化16HBE细胞中ERCC1基因表达和序列的变化

目的 探讨氯化镉(CdCl2)诱发人支气管上皮16HBE细胞恶性转化过程中,ERCC1基因mRNA和蛋白动态变化的规律及其序列变化情况.方法 用逆转录聚合酶链反应(RT-PCR)和免疫组织化学SP法,检测16HBE细胞及其CdCl2恶性转化细胞不同阶段(第5、15、35代细胞及成瘤细胞)ERCC1基因mRNA和蛋白的表达情况.用直接测序法,分析ERCC1基因第3、4外显子的序列情况.结果 随着转化代数的增加,ERCC1基因mRNA和蛋白的表达逐渐降低,至第35代细胞后表达明显下调,mRNA表达从16HBE的0.7028±0.0170下降到第35代细胞的0.3480±0.0453,蛋白表达强度从16HBE的8.40±1.34下降到第35代细胞的0.20±0.44,差异均有统计学意义(P＜0.01).ERCC1基因第4外显子在第1位点出现腺嘌呤(A)缺失,为移码突变.结论 镉致癌的分子机制可能与ERCC1基因水平的下调和突变有关.     

胃癌BRCA1基因甲基化与BRCA1 mRNA表达的关系研究

目的：探讨乳腺癌易感基因1（BRCA1）启动子CpG岛甲基化与BRCA1 mRNA表达的相关性及其意义。方法采用甲基化特异性PCR技术检测37例胃癌组织和对应癌旁组织及6例正常胃组织中BRCA1基因启动子CpG岛甲基化状态;采用逆转录PCR技术检测37例胃癌组织和对应癌旁组织及6例正常胃组织中BRCA1 mRNA 表达水平。结果胃癌组织中 BRCA1基因启动子 CpG 岛总甲基化率为48.6%（18/37）,显著高于癌旁组织[5.4%（2/37）]和正常胃组织[0.0%（0/6）],差异有统计学意义（χ2=17.541、5.021,P均〈0.05）。胃癌组织中BRCA1 mRNA阳性表达率为48.6%（18/37）,显著低于癌旁组织[94.6%（35/37）]和正常胃组织[100.0%（6/6）],差异有统计学意义（χ2=19.215、5.520,P均〈0.05）。BRCA1基因启动子CpG岛甲基化与BRCA1 mRNA表达成负相关（ r=-0.515,P〈0.05）。结论 BRCA1基因启动子CpG岛甲基化可能是导致BRCA1 mRNA失表达的主要原因之一。     

Epigenetic silencing of O6-methylguanine DNA methyltransferase gene in NiS-transformed cells

Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. To uncover the molecular mechanisms of nickel sulfide (NiS)-induced cell transformation, we investigated epigenetic alterations in a set of DNA repair genes. The silencing of the O(6)-methylguanine DNA methyltransferase (MGMT) gene locus and upregulation of DNA methyltransferase 1 (DNMT1) expression was specifically detected in NiS-transformed human bronchial epithelial (16HBE) cells. In addition, we noted epigenetic alterations including DNA hypermethylation, reduced histone H4 acetylation and a decrease in the ratio of Lys-9 acetylated/methylated histone H3 at the MGMT CpG island in NiS-transformed 16HBE cells. Meanwhile, we identified concurrent binding of methyl-CpG-binding protein 2, methylated DNA-binding domain protein 2 and DNMT1 to the CpG island of the MGMT promoter, demonstrating that these components collaborate to maintain MGMT methylation in NiS-transformed cells. Moreover, depletion of DNMT1 by introduction of a small hairpin RNA construct into NiS-transformed cells resulted in a 30% inhibition of cell proliferation and led to increased MGMT gene expression by reversion of the epigenetic modifications at the MGMT promoter region. MGMT suppression and hypermethylation at the CpG island of the MGMT promoter occurred 6 days after NiS treatment, indicating that epigenetic modifications of MGMT might be an early event in tumorigenesis. Taken together, these observations demonstrate that epigenetic silencing of MGMT is associated with DNA hypermethylation, histone modifications and DNMT1 upregulation, which contribute to NiS-induced malignant transformation.     

TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers

Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. The hypothesis that aberrant methylation plays a direct causal role in carcinogenesis hinges on the question of whether aberrant methylation is sufficient to drive gene silencing. To identify downstream targets of methylation-induced gene silencing, we used a human cell model in which aberrant CpG island methylation is induced by ectopic expression of DNA methyltransferase. Here we report the isolation and characterization of TMS1 (target of methylation-induced silencing), a novel CpG island-associated gene that becomes hypermethylated and silenced in cells overexpressing DNA cytosine-5-methyltransferase-1. We also show that TMS1 is aberrantly methylated and silenced in human breast cancer cells. Forty percent (11 of 27) of primary breast tumors exhibited aberrant methylation of TMS1. TMS1 is localized to chromosome 16p11.2-12.1 and encodes a 22-kDa predicted protein containing a COOH-terminal caspase recruitment domain, a recently described protein interaction motif found in apoptotic signaling molecules. Ectopic expression of TMS1 induced apoptosis in 293 cells and inhibited the survival of human breast cancer cells. The data suggest that methylation-mediated silencing of TMS1 confers a survival advantage by allowing cells to escape from apoptosis, supporting a new role for aberrant methylation in breast tumorigenesis.     

Frequent methylation-associated silencing of a candidate tumor-suppressor, CRABP1, in esophageal squamous-cell carcinoma

Epigenetic alterations and the resulting inactivation of tumor suppressor genes often contribute to the development of various cancers. To identify novel candidates that may be silenced by aberrant methylation in esophageal squamous-cell carcinoma (ESCC), we analysed ESCC cell lines by a recently developed method known as bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA), and selected candidates through BAMCA-assisted strategy. In the course of this program, we identified frequent CpG methylation-dependent silencing of the gene encoding cellular retinoic acid binding protein 1 (CRABP1) in our panel of ESCC cell lines. Expression of CRABP1 mRNA was restored in gene-silenced ESCC cells after treatment with 5-aza 2'-deoxycytidine. The DNA methylation status of the CRABP1 CpG island with clear promoter activity correlated inversely with expression of this gene. CpG methylation of CRABP1 was frequently observed in primary ESCC tissues as well. Restoration of CRABP1 expression in ESCC cells lacking the protein reduced cell growth by inducing arrest at G(0)-G(1), whereas knockdown of the gene in cells expressing CRABP1 promoted cell growth. Among 113 primary ESCC tumors, the absence of immunoreactive CRABP1 was significantly associated with de-differentiation of cancer cells and with distant lymph-node metastases in the patients. These results indicate that CRABP1 appears to have a tumor-suppressor function in esophageal epithelium, and its epigenetic silencing may play a pivotal role during esophageal carcinogenesis. Its expression status in biopsies or resected tumors might serve as an index for identifying ESCC patients for whom combined therapeutic modalities would be recommended.     

Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation-sensitive single-strand conformation analysis (MS-SSCA) and direct sequencing using bisulfite-modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p < 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT-expressing telomerase-positive samples and was absent in telomerase-negative samples (p < 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression.     

Aberrant methylation and silencing of ARHI,an imprinted tumor suppressor gene in which the function is lost in breast cancers

ARHI is a maternally imprinted tumor suppressor gene that maps to a site on chromosome 1p31 where loss of heterozygosity has been observed in 40% of human breast and ovarian cancers. ARHI is expressed in normal ovarian and breast epithelial cells, but ARHI expression is lost in a majority of ovarian and breast cancers. Expression of ARHI from the paternal allele can be down-regulated by multiple mechanisms in addition to loss of heterozygosity. This article explores the role of DNA methylation in silencing ARHI expression. There are three CpG islands in the ARHI gene. CpG islands I and II are located in the promoter region, whereas CpG island III is located in the coding region. Consistent with imprinting, we have found that all three CpG islands were partially methylated in normal human breast epithelial cells. Additional confirmation of imprinting has been obtained by studying DNA methylation and ARHI expression in murine A9 cells that carry either the maternal or the paternal copy of human chromosome 1. All three CpG islands were methylated, and ARHI was not expressed in A9 cells that contained the maternal allele. Conversely, CpG islands were not methylated and ARHI was expressed in A9 cells that contained the paternal allele of human chromosome 1. Aberrant methylation was found in several breast cancer cell lines that exhibited decreased ARHI expression. Hypermethylation was detected in 67% (6 of 9) of breast cancer cell lines at CpG island I, 33% (3 of 9) at CpG island II, and 56% (5 of 9) at CpG island III. Hypomethylation was observed in 44% (4 of 9) of breast cancer cell lines at CpG island II. When methylation of CpG islands was studied in 20 surgical specimens, hypermethylation was not observed in CpG island I, but 3 of 20 cases exhibited hypermethylation in CpG island II (15%), and 4 of 20 cases had hypermethylation in CpG island III (20%). Treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, could reverse aberrant hypermethylation of CpG island I, II and III and partially restore ARHI expression in some, but not all of the cell lines. Treatment with 5-aza-2'-deoxycytidine partially reactivated ARHI expression in cell lines with hypermethylation of CpG islands I and II but not in cell lines with partial methylation or hypomethylation of these CpG islands. To test the impact of CpG island methylation on ARHI promoter activity more directly, constructs were prepared with the ARHI promoter linked to a luciferase reporter and transfected into SKBr3 and human embryo kidney 293 cells. Methylation of the entire construct destroyed promoter activity. Selective methylation of CpG island II alone or in combination with CpG island I also abolished ARHI promoter activity. Methylation of CpG I alone partially inhibited promoter activity of ARHI. Thus, hypermethylation of CpG island II in the promoter region of ARHI is associated with the complete loss of ARHI expression in breast cancer cells. Other epigenetic modifications such as hypermethylation in CpG island III may also contribute to the loss of ARHI expression.     

OPCML gene promoter methylation and gene expression in tumor and stroma cells of invasive cervical carcinoma

To investigate the CpG island methylation and the mRNA expression of OPCML gene in patients with cervical carcinoma, we collected tumor and stroma cells from 36 invasive cervical carcinoma samples and 16 normal cervical tissues as well as Hela cells. Methylation specific PCR was used to detect promoter CpG island methylation status, and fluorescence quantitative RT-PCR was used to detection of OPCML gene expression. Our data showed that OPCML gene promoter methylation may play an important role in the carcinogenesis of cervical carcinoma and OPCML gene may be a cervical carcinoma-associated candidate TSG (tumor suppressor gene).     

Aberrant promoter CpG methylation and its translational applications in breast cancer

Breast cancer is a complex disease driven by multiple factors including both genetic and epigenetic alterations.Recent studies revealed that abnormal gene expression induced by epigenetic changes,including aberrant promoter methylation and histone modification,plays a critical role in human breast carcinogenesis.Silencing of tumor suppressor genes(TSGs) by promoter CpG methylation facilitates cells growth and survival advantages and further results in tumor initiation and progression,thus directly contributing to breast tumorigenesis.Usually,aberrant promoter methylation of TSGs,which can be reversed by pharmacological reagents,occurs at the early stage of tumorigenesis and therefore may serve as a potential tumor marker for early diagnosis and therapeutic targeting of breast cancer.In this review,we summarize the epigenetic changes of multiple TSGs involved in breast pathogenesis and their potential clinical applications as tumor markers for early detection and treatment of breast cancer.     

新疆哈萨克族食管癌survivin启动子区CpG岛甲基化状态及其临床意义

目的： 检测消化道肿瘤早期相关基因survivin mRNA在哈萨克族食管癌组织中的表达及其启动子区CpG岛甲基化状态,并进一步探究survivin启动子甲基化是否参与了食管癌的发生。方法：提取哈萨克族食管癌患者癌组织及远端无癌组织RNA,RT-PCR检测组织中survivin mRNA的表达水平,甲基化特异性PCR (methylation specific PCR,MSP)检测survivin启动子区CpG 岛的甲基化状态。结果：在20对食管癌组织标本中,癌组织中survivin mRNA阳性表达率 (95%)明显高于远端无癌组织 (65%) (P＜0.05)。癌组织及远端无癌组织中survivin基因启动子区CpG岛多为杂合型甲基化,且两种组织间甲基化状态无明显差异 (P＞0.05)。结论：survivin mRNA表达水平的增高在哈萨克族食管癌的发生、发展过程中起到了一定的作用,但其表达与启动子区CpG岛甲基化无关,提示甲基化并未直接调控该基因的表达并促进哈萨克族食管癌的发生发展。