人類食道癌組織p16, Rb 抑癌基因及端粒脢活性的研究及其與檳榔的關係

Betel nut & Human Esophageal Cancer- A Study of Telomerase, & Rb and p16 Tumor suppression gene

余方榮(Fang-Jong Yu)

指導教授 ： 王文明

高雄醫學大學/醫學院/醫學研究所/碩士(2003年)

摘要

摘要
目前台灣地區嚼食檳榔的人數逐漸增多，在動物實驗或人類的研究報告，檳榔是一種強力的致癌物；在動物實驗也證實檳榔可導致舌癌、口腔癌、食道癌、肝癌、肺癌及胃癌等。台灣本土的公共衛生研究發現，台灣2100萬人口中，約200萬人是現在或曾經嚼食檳榔的紅唇族。有關檳榔與食道癌的研究並不多，但是目前對於食道癌癌化過程模式是如何、檳榔是如何影響食道癌癌化過程，而抑癌基因P16及Rb變異方式及端粒脢和端粒脢活性對食道癌所扮演的角色之探討則更是少有報告。
P16基因位於9q21位置上，其基因蛋白質p16會與細胞周期所依賴的酵素cdk4結合，而阻斷細胞周期由G1期移行到S期。Rb基因位在染色體的13q14上，為一種110kDa的核蛋白，這種核蛋白可依被磷酸化的狀態，在細胞週期G1期的控制檢查點上表現和控制。
最近的研究發現，染色體末端的telomere(端粒)片斷長度和細胞能否長久存活，或驅向老化、死亡有關。而端粒長度的維持，有賴telomerase(端粒脢)這種酵素的作用，它藉著合成telomeric DNA來補充染色體末端的長度，造成細胞的癌化及不死化。人體內，端粒脢這種酵素在一般的體細胞是呈不活化狀態，因此一般體細胞均有一定的壽命，當它們經過一定數目的複製分裂，便會因為端粒太短而造成細胞不穩定狀態而死亡。然而端粒脢於一些腫瘤組織及不死化細胞株被發現有80%以上是活化狀態，是以體細胞內端粒脢這種酵素的激活化被認為可能是細胞癌化及不死化的重要因子。
本研究以人類食道癌進行研究，分為二部分，第一部分探討P16及Rb抑癌基因的突變情形，及分析檳榔、菸、酒是不是在其中扮演某種角色。本研究共有33位病人經檢查為食道癌病人（男31名，女2名，平均年齡57歲），取其開刀或內視鏡檢查時夾取之食道癌病變組織及正常組織，分別標示後馬上以液態氮冰凍並放入-80?C冰箱保存。經萃取DNA並經聚合脢連鎖反應(PCR)分析(p16)及聚合脢連鎖反應-單股結構多形性分析PCR-SSCP(Rb)。
第二部分共取24名 (男21名、女3名；平均年齡58.6歲) 經內視鏡檢查及病理組織切片檢查証實食道鱗狀細胞癌的病人，所有的病人在開刀後立即取出食道癌組織及週邊正常組織並分別抽取組織蛋白質，同時我們也取7名Barrett’s食道症病人的組織，應用TRAP(telomereic repeat amplication protocol)方法增幅，且用酵素免疫呈色法(ELISA)來測定食道癌或正常組織中端粒脢酵素的活性，同時也應用美國羅氏公司LightCycler聚合脢連鎖反應儀同步定量RT-PCR產物，分析食道癌組織中，端粒脢及端粒脢相關RNA(hTR，hTERT)表現的情形，並比較檳榔、菸、酒與食道癌之相關性。
結果發現:
1) 並未發現有P16基因不正常甲基化(methylation)之情形；而聚合脢連鎖反應(PCR)、聚合脢連鎖反應-單股結構多形性分析PCR-SSCP法探討Rb基因突變上也未發現有明顯的異常。故本研究第一部分結果顯示抑癌基因P16及RB基因在食道癌組織並無顯著變化及和煙、酒、檳榔間並無相關。
2) 端粒脢相關RNA(hTERT)之陽性率為83.3% (20/24)；而正常部分之陽性率為16.7% (4/24)(p<0.0001)呈有意義的差異。若分析病人抽煙、喝酒及嚼食檳榔等三個因素，其端粒脢相關RNA(hTERT)之相關與上述三個習慣有無來比較則發現沒有顯著的差異。(Logistic Regression test)
3) 在Barrett’s食道症這個癌前病變的七個病人當中，病灶區之端粒脢相關RNA(hTERT)之陽性率為85.7%(6/7)，而正常區的端粒脢相關RNA(hTERT)之陽性率為100% (7/7) (p= 0.6547)。
故本研究結果顯示抑癌基因P16及Rb基因在食道癌組織並無顯著變化及和煙、酒、檳榔間並無相關。以端粒脢相關RNA (hTERT)來表示端粒脢之活性結果發現在食道癌部分的活性顯著較正常部分來得高；而分析病人有抽煙、喝酒及嚼食檳榔等三個因素，其端粒脢相關RNA(hTERT)之相關與上述三個習慣的有無來比較則發現沒有顯著的差異。在Barrett’s食道症病人方面其病灶區之端粒脢相關RNA (hTERT)與正常部分則沒有顯著差異。所以端粒脢及端粒脢相關RNA (hTR，hTERT)在食道癌癌化過程中可能扮演著重要的角色。

關鍵字

檳榔 ； 食道癌 ； p16基因 ； Rb基因 ； 抑癌基因 ； 端粒脢

並列摘要

Abstract
The prevalence of betel quid chewing habit has been increasing in Taiwan area, and there were reports concerning about the relationships between betel quid and cancers, either in animal model or human, stated that betel quid was a potent carcinogens. It can lead to tongue cancer, oral cancer, esophageal cancer and even hepatoma, lung cancer & gastric cancer. Betel chewing enjoys island-wide popularity among the inhabitants of Taiwan; the number of current and ex-user was estimated at a 2.0 million people. However, there is no conclusion about the carcinogenesis of esophageal cancer, how the betel quid influence the carcinogenesis and what the role P16 and Rb tumor suppressor gene & telomerase are.
P16 gene and RB gene are tumor suppressor genes and studied in many cancers. The cyclin-dependent kinase inhibitor p16 gene, mapped on chromosome 9p21, is presumed to be the tumor-suppressor gene and that helps to stop transition from the late G1 to the S phase of the cell cycle. Rb gene is mapped on chromosome 13q14. Transition from the preproliferative G1 to the proliferative S phase of the cell cycle is regulated by phosphorylation of the retinoblastoma (Rb) protein.
Recently, telomeric length of chromosome ends has been implicated in the control of cellular immortality and aging. Telomeric DNA may be maintained by a specific enzyme: telomerase, which synthesize telomeric DNA repeats onto chromosome ends, thus stabilize the telomeric length. In human, somatic cells do not express telomerase activity. Therefore, most normal somatic cells have a strictly limited life span and undergo cell death on account of telomere shortening after a defined number of cell division. However, the telomerase activity has recently been found to present in over 80% of human tumors and immortalized cell lines. This indicates that activation of the enzyme telomerase is likely to be an important factor involved in tumorigenesis and cell immortalization.
In the first part of studies, we checked up p16 and Rb gene mutation in human esophageal cancer, and the role of betel nut and smoking and alcohol. 33 patients with esophageal cancer (31 men and 2 women, average age: 57y/o) were enrolled in this study. Collect tissue samples either from operation material or endoscopic tissue biopsy, including tumor and normal parts. Immersed tissue into liquid nitrogen immediately and than stored in -80?C. After isolation DNA from cancer tissue, We use polymerase chain reaction (PCR) method to check up P16 DNA methylation, and PCR and SSCP method to check up RB DNA mutation. In the second port of this studies, we collected 24 esophageal cancer patients (men： 21, female：3; mean age 58.6 y/o ) that is pathologically proved as esophageal squamous cell carcinoma by endoscopic examination or operation. We analyzed the telomerase activity and telomerase-associated RNA (hTR and hTERT) of cancer and surround normal tissue. We also collect 7 Barrett’s esophagus patients and analyzed the telomerase activity and its associated RNA (hTR and hTERT).
Our result showed that no significant p16 DNAmethylation nor RB gene mutation found. In the second port of studies, the positive rate of telomerase-associated RNA (hTERT) was 83.3% (20/24) in esophageal cancer part and 16.7% (4/24) in surrounding normal tissue. If compared with those who had smoking, alcohol drinking and betel quid chewing or not, there was no significant difference between substances use and positive rate of hTERT in esophageal cancer. Among those 7 patients who got Barrett’s esophagus, the positive rate of telomerase-associated RNA (hTERT) of the disease part and surrounding normal part was 85.7% and 100 % respective. ( p= 0.6547)
So we couldn’t found mutation pattern of P16 and RB gene. And we also couldn’t explain the role of betel nut chewing playing in esophageal cancer and p16 or RB gene mutation, and protein expression of P16 and RB, and the relationship with betel nut chewing, smoking and alcohol drinking. We also concluded that the telomerase- associated RNA (hTERT) may play an important role in the carcinogenesis of esophagus cancer.

並列關鍵字

Betel nut ； esophageal cancer ； p16 gene ； Rb gene ； tumor suppressor gene ； Telomerase

參考文獻

參考文獻

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