摘要
在自然环境中，天然辐射的50％来自于氡及其子体放射出来的α粒子，高水平的氡及其子体是诱发原发性肺癌的重要因素之一。对此，尽管人们已有深刻认识，但是时至今日氡及其子体诱发肺癌的分子机理仍未完全阐明。本实验室采用人支气管上皮细胞BEP2D建立了α粒子辐射致恶性转化的细胞模型，照射后第35代细胞接种于裸鼠皮下可形成高分化的鳞癌，继续分离培养获得了BERP35T1—BERP35T-6多株恶性转化的单克隆细胞，在国内率先开展了辐射致肺癌机制的研究。
蛋白质组水平的研究发现，BEP2D永生化细胞和BERP35恶性转化细胞中有多种蛋白质存在表达差异，其中MTAP、HMG-1在恶转细胞中表达降低，而Maspin在癌前阶段表达下降。在此基础上，我们以BEP2D细胞为对照，通过双向电泳技术对恶性程度更高的BERP35T-2细胞进行分析，并通过Northern杂交发现在BERP35T-2中MTAP的mRNA表达水平比BEP2D细胞中降低5倍以上。
MTAP(Methylthioadenosine Phosphorylase，甲硫腺苷磷酸化酶)，是甲硫氨酸和嘌呤合成补救途径的第一个酶，专门司职催化多胺代谢的副产物MTA(Methylthioadenosine，甲硫腺苷)，使之生成腺嘌呤和甲硫核糖-1-磷酸(5-methylthio-D-ribose 1-phosphate，MTR-1-P)，最后形成ATP、dAMP和甲硫氨酸，分别参与细胞的能量合成、DNA合成和蛋白质合成。MTAP催化的是可逆反应，在正常细胞中有非常重要的作用。
关于MTAP基因在肺癌或BEP2D辐射致癌细胞模型中的研究，在国内并无相关报道，这促使我们围绕MTAP基因组及其邻近基因(组)结构在辐射致癌模型和原发性非小细胞肺癌中展开了调查研究，主要取得了以下几个方面的结果：
1．在细胞模型中，MTAP基因在辐射诱发支气管上皮BEP2D细胞恶性转化过程中表达显著降低，蛋白质水平和转录水平差别均在5倍以上。
军事医学科学院卫生毒理学专业博士学位论文2003年6月
SSCP方法检测发现MTAP基因在BERP35T-2中没有发生突变;PCR分析
也没有纯合性缺失和启动子异常甲基化现象;但MTAP基因组附近的微卫
星位点RH99034发生了杂合性缺失(1055 of heterozygosity，LOH)，这可能
是表MTAP达下调的原因。这些结果提示该基因可能与细胞的恶性转化有
关。
2.Ml，AP基因在非小细胞肺癌中的纯合性缺失研究。采集了44例原
发性非小细胞肺癌，用PCR一ELISA方法分析，发现有9.1%的病例(4/44)
MTAP基因发生了纯合性缺失。而据文献报道经常与之高比例共缺失的
P16基因，只检测到6.8%的(3/44)纯合性缺失率。提示低频率的纯合性
缺失可能是Ml，AP在肺癌中失活的原因之一。
3.MTAP基因启动子异常甲基化和突变分析。采用MSp(Methylation
一sPecific PcR)原理针对MTAP启动子设计了引物，对44例非小细胞肺
癌的基因组DNA处理后进行PCR扩增。发现MTAP启动子异常甲基化的
比例为6.8%(3/44)，说明启动子异常甲基化可能是MTAP基因失活的因
素之一。SSCP分析发现Mrl人P在非小细胞肺癌中没有发生碱基突变。这
说明MT人P基因表达调节可能不是靠碱基突变方式控制的。
4.MTAP在非小细胞肺癌中的杂合性缺失研究。在MTAP基因组附近
选取了7个STS标志:D95162、D951749、D95916、RH99034、D951748、
D951752和D95977。对之进行分析，发现7个STS的LOH频率分别为:
1 3.8%(5/36)、56.2%(18/32)、54.2%(17/33)、70.3%(19/27)、57.1
%(16/28)、30.8%(8/26)和13.3%(4/30)。其中44例标本中61.9%(26/42)
至少有一个STS位点发生LOH现象。中低分化肺癌中LOH比例显著高于
高分化肺癌。这些结果说明gpZI区域高频率的LOH现象可能是导致MTAP
基因表达变化的主要原因，并且与肿瘤的分化程度有关。此现象提示Ml，AP
在非小细胞肺癌中表达降低或丢失可能独立于pl6缺失，说明其缺失或低
表达可能有其肿瘤生物学基础。
5.Ml，A.P在非小细胞肺癌中翻译和转录水平的验证。选用了15例新
鲜肺癌标本，进行RT-PCR分析，发现Ml，AP在73.3%(11/15)的肿瘤标
本中不表达或转录水平降低5倍以上，而MJTAP在正常对照组织中的表达
率却高达93.3%(14/15)。对n例Rr‘PCR分析认定低表达或不表达的样
军事医学科学院卫生毒理学专业博士学位论文2003年6月
本，用Western Blot方法进一步验证，发现与RT‘PCR结果一样，基本与
在基因组水平分析的结论一致。MTAP在肺腺癌和鳞癌中低表达现象无显
著差异，但在中/低分化肺癌中低表达率显著高于高分化肺癌，再次提示
MTAP基因低表达或丢失可能与肿瘤的恶性程度密切相关。
6.研究MTAP的同时，本研究分析了与MTAP基因连锁的P16、P14
和P15等抑癌基因在肿瘤标本中转录水平的变化。RT.PCR方法分析，发
现与正常组织相比，P15在7例肺癌标本中有5例基本不表达;在7例标
本中，意外地发现P16和P14在正常组织中转录水平很低，RT.pCR方法
基本无法检测到相应cDNA，而在配对的肺癌组织中却分别有3例高表达。
这提示MTAP的缺失可能是独立于pl6之外的，其低表达或缺失在肿瘤生
物学中并不是偶然现象，可能有其特殊的生物学意义等待发掘。
本研究的创新?
5'-Deoxy-5'-methylthioadenosine phosphorylase (methylthioadenosine: ortho- phosphate methylthioribosyltransferase, EC 24.2.28) plays a key role in purine and polyamine metabolism and in the regulation of transmethylation reactions. MTAP is abundant in normal cells but deficient in many kinds of tumors and tumor-derived cell lines. MTAP is located on human chromosome 9p21, where tumor suppressor genes such as pi6, p!4ARF and pi5 also map. It is believed that MTAP is frequently codeleted with pi6.
By 2-Dimension Electrophoresis analysis, MTAP was observed to be down-regulated in the BERP35 cells originated from BEP2D cells irradiated by a single dose of high-energy a particles. To investigate the association of down-regulation of MTAP gene with malignancy of cells passaged from BERP35, total proteins from BEP2D cells and BERP35T-2 derived from BERP35, which shows further malignancy, were extracted and analyzed by 2-D electrophoresis. MTAP was found to be expressed at very low level in BERP35T-2 cells somewhat similar to BERP35.
Northern Blot and Western Blot were performed respectively to confirm differential expression of MTAP gene between BEP2D and BERP35T-2. MTAP was found to be expressed 5 times lower at mRNA level as well as protein level in BERP35T-2 than in BEP2D.
Genomic DNA was extracted from cell lines BEP2D, BERP35T-2 and 44
cases of Chinese non-small cell lung cancers. No homozygous deletions and mutations were detected in BERP35T-2 cells, and no methylation was detected in the CpG island of the promoter of MTAP gene by Methylation-Specific PCR. Three STSs, D9S171, D9S162 and RH99034, flanking MTAP gene region, were analyzed by PCR amplification, and LOH (loss of heterozygosity) was found at RH99034 locus.
Genomic alterations were investigated in 44 samples. Homozygous deletions of MTAP were detected in 4 of 44 NSCLC samples. No Mutation was found in all cases by SSCP. The CpG island in the promoter of MTAP gene was methylated in 3 out of 44 cases detected by MSP. Seven microsatellite loci, D9S162, D9S1749, D9S916, RH99034, D9S1748, D9S1752 and D9S977, distributing from 9p21 to 9p22, were selected to investigate frequency of LOH in 44 NSCLC samples. LOH frequencies of the seven STSs markers were 13.8%(5/36), 56.2%(18/32), 54.2%(17/33), 70.3% (19/27), 57.1%(16/28), 30.8%(8/26) and 13.3%(4/30), respectively. 26 of 42 informative samples showed at least one locus lost.
15 fresh samples of the 44 cases were analyzed for MTAP mRNA and protein expression by RT-PCR and Western Blot. Down-regulation or loss of MTAP expression was found in 11 cases, showing consistency with the alterations occurring at genomic level. Furthermore, RT-PCR was performed to observe the differences between MTAP gene and tumor suppressor genes pi6, p!4 and pi5. Unexpectedly, it was found that pi6 and pi4 were expressed at higher level in 3 of 7 tumor samples respectively than in matched normal tissues, while pi5 mRNA was deficient in 5 tumor samples.
The results indicate that down-regulation of MTAP in BERP35T-2 cell line caused by LOH at RH99034 may contribute to the elevating malignancy of BERP35T-2. LOH occurred at 9p21 genomic region in Chinese NSCLC may contribute more to deficiency or down-regulation of MTAP than homozygous deletions and methylations of the only CpG island in the promoter region.
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