行政院國家科學委員會專題研究計畫 期中進度報告
以 LMS/CGH/LOH 等技術來研究檳榔相關口腔癌及癌前變 化之基因變異/粒腺體傷害與臨床預後之相關性(2/3)
計畫類別： 個別型計畫
計畫編號： NSC91-2320-B-006-057-
執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 國立成功大學醫學系病理學科
計畫主持人： 靳應臺
報告類型： 精簡報告
處理方式： 本計畫可公開查詢
中 華 民 國 92 年 5 月 14 日
行政院國家科學委員會補助專題研究計畫 期中進度報告
以 LMS/CGH/LOH 等技術來研究檳榔相關口腔癌及 癌前變化之基因變異/粒腺體傷害與臨床預後之相 關性
計畫類別： 個別型計畫
計畫編號：NSC 91 －2320 －B －006 －057 執行期間：91 年 08 月 01 日至 92 年 07 月 31 日 計畫主持人：靳應臺
成果報告類型(依經費核定清單規定繳交)：精簡報告
執行單位：成功大學醫學院病理學科
中 華 民 國 92 年 05 月 15 日
Abstr act
Oral cancer is the fourth leading cause of cancer deaths among men in Taiwan and is closely association with areca quid chewing habits. Recent studies showed that mitochondrial DNA (mtDNA) mutations occur in various tumors including oral cancers and that the accumulation of mtDNA deletions could be an important
contributor during carcinogenesis. We have analyzed mtDNA deletions by pairwise comparisons among oral cancer, precancerous cells and their adjacent submucosal stoma tissues in 12 patients with areca quid history using laser microdissection.
Real-time quantitative PCR (RTQPCR) was performed to detect and quantify mtDNA with the 4977 bp deletion in the histologically defined specified cell groups.
Quantitative analysis of 60 samples by RTQPCR revealed that the average
proportions of 4977 bp-deleted mtDNA over total mtDNA were 0.137%, 0.367 %, and 0.001% in cancer, precancer cells, and lymphocytes of lymph node biopsies, respectively. Pairwise analysis of the proportion of mtDNA deletion in the cancer, precancer and their stoma tissues revealed a consistent trend among these patients. All of the patients (12/12) presented higher proportion of mtDNA with 4977 bp deletion in the lesions than that in the lymphocytes, with an average increases of 198 folds in cancer, and 546 folds in precancer cells. A decrease in the proportion of deleted mtDNA was observed in 8 of 12 patients when the disease progressed from precancer to cancer lesions. Interestingly, 7 out of 12 cancer tissues and 8 out of 12 precancer lesions exhibited an average of 6.3-fold and 17.4-fold increases in the proportion of 4977 bp-deleted mtDNA in the stromal cells than in the lesion cells, respectively.
The observation that the proportion of 4977 bp-deleted mtDNA in all oral lesions was higher than normal and consistently decreased during cancer progression from
precancer to primary cancer suggests that accumulation and subsequent cytoplasmic segregation of the mutant mtDNA during cell division may play an important role in oral carcinogenesis. This study also demonstrated that laser microdissection
combined with RTQPCR is an efficient and unequivocal approach to gain insight into the role that mtDNA mutation may play in carcinogenesis.
Keywords: Real-time quantitative PCR, laser microdissection, Mitochondrial DNA deletion, mouth neoplasm
中文摘要
在台灣，口腔癌死亡率已躍進成十大死亡癌症的第四位，且這些口腔癌患者大 部分皆有嚼食檳榔的習慣。 近年來的研究指出，在許多癌症（包括口腔癌）皆 發現有粒腺體 DNA(mtDNA)變異存在，且這些粒腺體 DNA(mtDNA)變異的累積 很有可能是一個重要的致癌因子。 我們先利用雷射顯微切割儀（LMD）收集 12 位有嚼食檳榔口腔癌患者個人癌前變化、腫瘤和其周圍間質的組織。使用即時定 量聚合脢連鎖反應(RTQPCR)分析這些不同變異組織中 4977bp depleted mtDNA 的比例。在分析的 60 檢體當中，平均 4977bp depleted mtDNA(除以總 mtDNA)
的比例分別為腫瘤組織(0.137%)、癌前變化組織（0.367%）、淋巴組織（0.001%）。
我們發現在這 12 位患者中 4977bp depleted mtDNA 在不同組織的比例皆有一致 性的趨勢，所有患者（12/12）癌前變化和腫瘤組織的 4977bp depleted mtDNA 比 例皆高於淋巴組織(546 倍、198 倍)。在口腔癌癌過程中，發現八個患者(8/12) 癌 前變化組織中的 4977bp depleted mtDNA 比例比腫瘤組織高。有趣的是，7/12 腫 瘤組織和 8/12 癌前變化組織其間質組織有較高 4977bp depleted mtDNA 比例 (6.3 倍、17.4 倍)。 所以我們推測 mtDNA 突變的累積和細胞分裂時 mtDNA 在細胞 質中的分離可能在口腔癌癌過程中扮演一個重要的角色。這個實驗也證明了雷射 顯微切割儀(LMD)結合即時定量聚合脢連鎖反應(RTQPCR)是一個有精確且效率 研究方法可用來研究癌化過程中的粒腺體 DNA 的突變。
Introduction
Oral cancer is the fourth leading cause of male cancer death and in Taiwan attributed to the prevalent areca quid chewing habits.1 Previous studies have shown that ingredients of areca quid generate significant amount of reactive oxygen species (ROS) and induce oxidative DNA damage in cultured cells.2, 3 Although a wide spectrum of genomic alterations in nuclear DNA in carcinogenesis has been
established, little attention has been paid to the role of mitochondrial DNA (mtDNA) mutations in human cancers. Human mitochondrial genome is composed of a circular double stranded DNA of 16.6 kb, which encodes 13 polypeptides constituting the respiratory enzyme complexes, 22 tRNAs and 2 rRNAs in the organelle. There may be hundreds to thousands of mitochondria in one cell and each may contain two to ten copies of mitochondrial DNA. The ROS-induced mtDNA damage is usually accumulated in somatic tissues and more likely reflects the exposure history than the nuclear DNA as mitochondrial genome is more susceptible to environmental insults for a relatively unprotected DNA structure and less sophisticated DNA repair mechanism.4, 5 Moreover, the large number of copies and the existence of
heteroplasmy of the mitochondrial genome have enabled the affected cells to continue survive with the damaged mtDNA, which may quantitatively reflect the extent of exposure to ROS.6
Different types of mtDNA mutations have been shown to play important roles in human aging and many metabolic, degenerative or neuromuscular diseases.5
Recently, somatic mutations of mtDNA were found to occur in various types of human cancers including colorectal carcinoma, breast, thyroid, and gastric cancers.7-12 The observation that most of these mutations are homoplasmic indicates the
advantage of the mutant mtDNA towards malignant progression. Although a number of point mutations in mtDNA have been identified in human tumor tissues, only few research groups have investigated the presence and accumulation of mtDNA deletions in cancers. Using semi-quantitative PCR analysis, Lee et al.13 was the first to demonstrate a significant association between areca quid chewing habit and the occurrence of 4977 bp mtDNA deletion in paired oral cancer tissues. Interestingly, a drastic increase of mtDNA deletion was observed in the stoma tissue than the tumor tissue in both groups with or without areca quid history. Consistent finding that stromal cells harbored a higher proportion of mtDNA with 4977 bp deletion than the tumor counterparts was also reported by Tan et al.14 in a study of 18 oral cancer tissues.
Here we report a new approach toward a more comprehensive quantitative analysis of mtDNA mutations in histologically well-defined cell populations utilizing a combination of laser microdissection (LMD) and real-time quantitative PCR
(RTQPCR) analysis. The proportion of mtDNA with 4977 bp deletion was analyzed by RTQPCR in the microdissected peripheral lymph node, precancer, cancer cells and their surrounding stromal cells that were isolated from 12 patients with a history of sequentially developed oral lesions
.
Results
Accumulation of 4977 bp deleted mtDNA in the paired laser microdissected tissues during oral carcinogenesis progression. Indicated tissue cells of the same individual were analyzed using real-time quantitative PCR. As expected, the proportion of the 4977 bp-deleted mtDNA in the lymph node was significantly lower than the DNA isolated from all other types of dissected samples (p<0.05). The mean proportion of the 4977 bp-deleted mtDNA was the highest in the precancer stroma, followed by precancer, cancer stroma, and cancer.
