細胞週期調節基因在維生素甲酸誘發人類肝癌細胞株凋亡時之表現

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Title:	細胞週期調節基因在維生素甲酸誘發人類肝癌細胞株凋亡時之表現
Other Titles:	Expression of Cell Cycle Regulatory Genes in Retinoic Acid-Induced Apoptosis in Human Hepatoma Cell Line
Authors:	陳美智
Contributors:	黃光裕;徐士蘭 Hwang, Guang-Yuh;Hsu, Shih-Lan 東海大學生命科學系
Keywords:	蛋白質激脢抑制分子;蛋白質激脢;細胞凋亡;維生素甲酸 p27);CKIs (p21;CDKs;apoptosis;retinoic acid
Date:	1997
Issue Date:	2011-06-15T03:38:58Z (UTC)
Abstract:	本論文以人類肝癌細胞株 Hep3B 為研究模式，探討維生素甲酸 (Retinoic acid ；簡稱 RA) 誘發細胞凋亡的過程中，是否與細胞週期調節基因的異常表現有關？結果發現在無血清的情況下，處理 RA 會造成 Hep3B 細胞死亡，尤其在 10^-5 M 高濃度的處理下細胞死亡率近乎 100 %；而由細胞螢光染色和 DNA 斷片電泳分析等結果證實 RA 誘發細胞進行凋亡。從細胞回復能力的實驗得知，連續處理 RA 8 小時後細胞存活比率為 13 %，24 小時後則為 2.27 %，此結果可推斷在凋亡過程中，於 24 小時之前產生變化的分子可能扮有重要的調控角色。此外，細胞分析儀 (flow-cytometer) 的分析結果亦證實 RA 影響細胞週期分佈。由蛋白質電泳及激脢活性的分析結果得知，在凋亡過程中，某些細胞週期調節基因的表現的確發生變化：Hep3B 細胞經 RA 處理後，CDK1/CDC2 的蛋白質表現漸增，其活性也受 RA 影響而增強；CDK2 蛋白質電泳結果則顯示經 RA 處理後，磷酸化之 CDK2 逐漸減少，其活性則在 4 小時被明顯的抑制。經處理 RA 8 小時，蛋白質激脢抑制分子 CIP1 (p21)的表現明顯增加，本論文並證實 RA 是經由轉錄( transcription ) 的層次增加 CIP1 的產量。 In this article, we used human hepatoma Hep3B cell line to examine molecular mechanism of the RA-induced cell death. Acridine orangestaining and DNA fragmentation analysis were performed to determinewhether RA-induced cell death undergo the apoptotic process. Our resultsindicated that treatment with RA in serum free condition lead to apoptosis.Time course studies of RA addition into the culture showed that the viabilityof Hep3B is approximate 13 % after RA treatment, and less than 3 % after24 h of RA treatment. This result implicated that the critical regulatorswhich involved in RA-induced apoptosis must be activated before 24 h.Since flow cytometric analysis revealed that RA can modulated cell cycledistribution during apoptotic process. We subsequently analyzed the expression of cell-cycle regulatory molecules to investigate the roles ofthese molecules in apoptosis. Western blot analysis and kinase activity assay were carried out to detect the effect of RA on these molecules.following RA treatment, the protein levels of CDK1/CDC2 was slightlyincreased, and associated with a marked increasing in activity. At thesame treatment, RA also altered the expression of CDK2, with a gradual disappearance of the upper band. However, the kinase activity of CDK2 was significantly inhibited at 4 h. Additionally, we also found RA treatmentactivated the expression of CIP1(p21) both on protein and RNA levels.
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