p21蛋白和HPV感染對Rb蛋白消失在台灣地區肺癌之角色探討

中山醫學大學機構典藏 CSMUIR > 醫學院 > 醫學分子毒理學研究所 > 博碩士論文 > Item 310902500/1003

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Title:	p21蛋白和HPV感染對Rb蛋白消失在台灣地區肺癌之角色探討 The role of p21 protein and HPV infection on Rb protein inactivation in Taiwanese lung tumorigenesis
Authors:	陳培亮 Pei-Liang Chen
Contributors:	中山醫學院：毒理學研究所;李輝
Keywords:	視網膜腫瘤;非小細胞肺癌;人類乳頭狀瘤病毒 Rb;p21;waf1;cyclin D1;HPV
Date:	1999-06-08
Issue Date:	2010-03-25T09:01:49Z (UTC)
Abstract:	過去的研究顯示 p53 基因發生突變是引起肺癌的重要致病原因。但台灣地區非小細胞肺癌 (non-small cell lung cancer, NSCLC) 的 p53 突變頻率 (18%) 較過去的報告都低 (50-80%)，這顯示台灣地區肺癌發生的機轉可能有其他更重要的基因參與。本研究假設 Rb 基因可能參與台灣肺癌的腫瘤化機轉。因此本研究先利用免疫組織化學染色法分析 247肺癌病患肺腫瘤的 Rb 表現。結果發現有 68.4% 的患者 (169/247) 無法偵測到 Rb 蛋白的表現。為了探討 Rb 蛋白消失的原因，我們推測可能是 (1)參與 Rb 蛋白的磷酸化基因，例如 p21、cyclins 和 cyclin dependent kinase 等蛋白表現不正常所致。 (2) 感染乳頭狀瘤病毒而將 Rb 蛋白分解。 (3) Rb 基因的轉錄層次受損。首先同樣以免疫染色法分析 p21 和 cyclin D1 的蛋白表現，結果發現有將近一半的病患 (45.7%, 113/247) 無法偵測到 p21 蛋白的表現。同時發現 p21蛋白與 Rb 蛋白的表現呈顯著正相關 (p=0.008)。同樣地，cyclin D1蛋白的表現也與Rb蛋白表現呈顯著相關 (p<0.001) 。這些結果顯示 Rb 蛋白的消失可能是 p21 無法有效抑制 Rb 蛋白磷酸化所造成。其次以 nested PCR 偵測肺癌組織是否有乳頭狀瘤病毒的 DNA，以確定肺癌患者是否感染乳頭狀瘤病毒。令人訝異的是竟然發現有 55.2% 的肺癌患者感染乳頭狀瘤病毒，此感染率明顯高於其他國家曾經發表過的相關報告(0-36%)，因此認為人類乳頭狀瘤病毒的高感染率可能經由誘導ubiquitin-proteasome 路徑，將肺腫瘤組織中 p21 蛋白與 Rb 蛋白迅速分解。可能在台灣地區肺癌肺腫瘤化過程有相當程度的關係。另外利用 RT-PCR 和 genomic PCR來分析 Rb 基因的轉錄層次是否發生改變，結果發現僅有 16-19% 的 Rb 蛋白消失是由基因轉錄機轉缺陷所致。在臨床上預後研究上，發現 Rb 蛋白表現僅在 p21 蛋白消失的患者才能作為預後指標 (P = 0.0078)，即 Rb/p21 同時消失的肺癌患者之存活時間顯著短於 Rb+/p21- 的患者。由以上的結果可知肺癌患者 Rb 蛋白消失可能和 p21 蛋白表現異常及人類乳頭狀瘤病毒的高頻率感染有關。同時 Rb 蛋白消失在台灣地區肺癌腫瘤機轉上可能扮演重要的角色 In this study, we propose that Rb protein inactivation may play a role on Taiwanese lung tumorigenesis, because relatively lower frequency of p53 gene mutation (18%) was found compared with previous data reported in other countries (50-80%). To test the hypothesis, immunohistochemical analysis was used to evaluate the expression of Rb protein in 247 primary non-small-cell lung cancer (NSCLCs) specimens. High frequency of Rb protein was undetectable by immunostaining (169/247, 68.4%). We suspect that the high frequency of Rb protein inactivation may be caused by (1) alteration of genes involved in Rb protein phosphorylation, such as p21, cyclins and CDKs, (2)inactivation by other oncoproteins from HPV infection, and (3) alteration of Rb gene transcription. Thus, we provide the following evidence to demonstrate which mechanism was important in Rb protein inactivation. Therefore, we first analyzed the protein expressions of p21 and cyclin D1 using immunohistochemistry. We found that high frequency of p21 negative immunostaining (113/247, 45.7%) and its expression was significantly associated with Rb protein expression (P = 0.008). Cyclin D1 protein expression was also correlated with Rb protein expression (P < 0.001). These results suggest that Rb protein inactivation may be caused by the increased its phosphorylation. Secondly, genomic nested PCR was used to detect whether HPV DNA was existed in lung tumor. Surprisingly, 55.2% (32/58) of HPV-18 DNA was detected in lung tumors. The HPV infection frequency in lung cancer found in this study was significantly higher than previous reports (0-36%). We thus strongly suggest that high HPV infection may partly result in the absence of p21 and Rb proteins through the induction of ubiquitin-proteosome pathway. Finally, the defect of Rb gene transcription was examined by reverse transcription PCR (RT-PCR) with the primer which was used to hybridize exon 2 of Rb gene and we used genomic PCR to examine whether promoter site of Rb gene was defected to cause down-regulated transcription. Our results showed that Rb protein inactivation were only 16-19% of cases to be explained by the defect of Rb gene transcription. In clinical outcome, the survival rate of patients with the p21 negative/Rb negative immunostaining was significantly shorter than patients with p21 negative/Rb positive (P = 0.0078). This finding further support that p21 negative immunostaining plays an important role on Rb protein inactivation. From the above findings, we strongly suggest that high frequency of Rb protein inactivation caused by p21 gene alterations and HPV infection may play an important role on Taiwanese lung tumorigenesis.
URI:	http://140.128.138.153:8080/handle/310902500/1003
Appears in Collections:	[醫學分子毒理學研究所] 博碩士論文

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