臺灣博碩士論文加值系統

肺癌為國人癌症死亡的首位，近年來每年約有6000人死於肺癌，但其分子致癌機制至今仍未釐清。由於癌症的形成過程中主要變異的基因為致癌基因 (Oncogenes) 活性過增或抑癌基因 (Tumor suppress genes) 失去活性；抑癌基因經研究證明需二個基因座皆變異才導致其失去活性而致癌，其變異的方式多為一基因座產生點突變、小片段鹼基缺失、或啟動子過度甲基化 (promoter hypermethylation)，而另一基因座產生抑癌基因及鄰近區域DNA大片段的缺失 (loss of heterozygosity)。本研究的目的在探討位於染色體3p14.2 的FHIT抑癌基因，在台灣地區非小細胞肺癌病人細胞中之變異情形。利用免疫組織化學染色法，觀察病人組織切片中Fhit 之蛋白質表現，結果顯示有50.4% (65/129) 非小細胞肺癌病人細胞中Fhit蛋白質表現量異常降低；免疫組織化學染色的結果亦發現在69% (47/68) 鱗狀上皮細胞肺癌 (squamous carcinoma) 病人中Fhit蛋白質表現有異常，顯示Fhit蛋白質異常在鱗狀上皮細胞肺癌形成過程中扮演一個重要的角色。利用反轉錄-聚合酵素鏈反應分析癌組織中mRNA轉錄是否異常，結果顯示有38.8% (38/98) 非小細肺癌病人細胞中FHIT mRNA轉錄有異常降低的情形，其中24位病人其反轉錄-聚合酵素鏈反應產物有長度異常的片段，經定序分析後顯示這些長度異常的片段多含FHIT基因exons 4-9的缺失，研判多源自mRNA剪接 (splicing) 錯誤所導致。利用亞硫酸基因定序分析偵測FHIT基因5’ CpG島叢甲基化情形，結果顯示有30.8% (28/91) 病人DNA 有高度甲基化的情形。由於免疫組織化學染色結果顯示出53% (35/66) 早期非小細胞肺癌病人其Fhit蛋白質有表現異常的情形；從12個非小細胞肺癌病人早期支氣管表皮細胞的分析中，亦發現50% (6/12)的病人支氣管表皮細胞中有FHIT基因甲基化的情形，此12個病人支氣管表皮細胞FHIT基因甲基化情形與其腫瘤組織之甲基化情形達83% (10/12) 之一致性，這些結果顯示FHIT基因甲基化在肺癌形成早期即發生。從FHIT基因甲基化、mRNA表現異常及蛋白質表達異常的相關性分析中，顯示彼此間皆有顯著相關 (P £ 0.0001)，且其中同時做了此三種不同分析的81人中，發現有60% (49/81) 的病人至少存有一種FHIT基因/蛋白質的變異。此實驗結果充分顯示出FHIT基因變異在台灣肺癌的形成過程中的重要性，未來可提供作為一個肺癌早期偵測的指標。

Lung cancer is the leading cause of cancer deaths in Taiwan. However, the molecular mechanisms involved in lung tumorigenesis in Taiwan remain poorly defined. It has been shown that alterations of tumor suppressor genes (TSGs) involve in the multi-step carcinogenesis of human cancer including lung cancer. Both copies of TSGs have to be inactivated for their function to be lost. One allele maybe inactivated by point mutation, methylation changes, or small deletions. The other allele is frequently inactivated by loss of heterozygosity, which showed a large deletion involving the gene of interest as well as adjacent stretches of DNA. The purpose of the present study was to investigate the etiological association of the FHIT tumor suppressor gene in lung tumorigenesis in Taiwan using the genetic and epigenetic analyses. The alteration of Fhit protein expression was examined by immunohistochemistry analysis. The results showed that 50.4% (65/129) NSCLC patients in Taiwan had decreased or loss expression of Fhit protein. Abnormal Fhit expression was more frequently found in squamous carcinomas (SQ, 69%) than in adenocarcinomas (AD, 28%) (P < 0.0001), suggesting that the FHIT alteration may play a significant role in the SQ lung tumorigenesis. Reverse transcriptase-polymerase chain reaction analysis of mRNA expression showed that, 38.8% (38/98) of patients exhibited FHIT transcripts with aberrant sizes or had low or absent FHIT mRNA expression. Sequencing analysis of the transcripts with aberrant sizes indicated that exons missing due to the splicing alterations were frequently detected. Methylation-specific PCR and bisulfite sequencing were conducted to detect the promoter methylation of the FHIT gene. The results showed that, 30.8% (28/91) of patients exhibited FHIT gene hypermethylation at exon 1 region. Decreased Fhit protein expression was detected in 53% (35/66) of tumors from stage-I and stage-II patients. In addition, the DNA of the bronchial epithelium cell from 12 lung cancer patients showed 50% (6/12) of methylation in the FHIT gene, and had an 83% (10/12) concordance of methylation with their tumor tissues. The data suggested that the FHIT gene and/or protein were altered in early lung tumorigenesis. Among a subset of 81 cases in which we were able to analyze all different types of FHIT aberration status, 60% (49/81) patients harbored at least one type of molecular alteration within the gene, strongly suggesting the importance of the FHIT alterations in NSCLC tumorigenesis. The results indicated that alteration of the FHIT gene might provide as a possible molecular marker for detection/diagnosis of NSCLC.

ABSTRACT ----------------------------------------------------------------- 1
INTRODUCTION ------------------------------------------------------- 5
I. The FHIT (fragile histidine triad) gene -------------------------- 6
II. Fhit protein structure and function -------------------------------- 7
III. Is FHIT a tumor suppressor gene? -------------------------------- 8
IV. FHIT gene/protein alterations in human cancers --------------- 9
V. FHIT gene/protein alterations in lung cancer -------------------- 10
VI. Mechanisms involved in FHIT alteration ----------------------- 12
PURPOSE ----------------------------------------------------------------- 15
MATERIALS AND METHODS ---------------------------------- 16
I. Tumor specimens and clinical characterization of patients----- 16
II. Microdissection and DNA extraction ------------------------- 17
III. Immunohistochemistry assay ----------------------------------- 18
IV. Multiplex RT-PCR analysis ------------------------------------- 19
V. cDNA sequencing ------------------------------------------------ 20
VI. MSP (methylation specific PCR) & bisulfite-sequencing
methods ------------------------------------------------------------ 20
VII. Multiplex PCR analysis ----------------------------------------- 22
VIII. Statistical analysis ----------------------------------------------- 23
RESULTS ------------------------------------------------------------------ 25
I. Fhit protein expression in NSCLC ------------------------------ 25
II. FHIT mRNA expression in NSCLC ---------------------------- 26
III. FHIT promoter hypermethylation in NSCLC ------------------ 28
IV. Correlation of FHIT methylation, mRNA expression,
and protein expression --------------------------------------------- 30
DISCUSSION ------------------------------------------------------------ 32
REFERENCES --------------------------------------------------- 39
FIGURES ------------------------------------------------------------------ 50
TABLES -------------------------------------------------------------------- 62

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