研究PERP (p53 apoptosis effect related to PMP-22) 基因對人類肺癌細胞株生長的影響

PERP(p53 apoptosis effect related to PMP-22)蛋白是在細胞膜上穿膜四次的膜蛋白，基因位置在染色體6p24，此區域是黑色素瘤、子宮頸癌、卵巢癌、乳癌常發生loss of heterozygosity (LOH) 的位置。因與PMP-22蛋白家族同源性高，故歸類為PMP-22家族的一員。過去報導指出，PERP在不同的細胞內，會受到p53或p63的調控，而扮演著不同的腳色。在MEFs細胞內，主要是受到p53調控，引發細胞走向凋亡。而在上皮細胞內，則是受到p63所調控，其功能則是影響上皮細胞的發育及分層。我們利用PERP抗體進行IHC，分析肺癌的tissue array, 發現有83%的肺腺癌及98%的鱗狀上皮癌罹過度表現PERP。在肺癌細胞株中， H1299、H1355、CL1-5及A549 PERP的mRNA表現量高，而CL1-0、H661的PERP mRNA表現量低。我們由A549及H1299細胞中得到十三個PERP cDNA clones，發現大部分的cDNA clones在538的位置上有一個missense突變，而只有一個clone是屬於wild type的序列。因此，我們在CL1-0細胞中過度表現wild type PERP及有538 mutation 的PERP基因，並在A549及CL1-5抑制PERP基因的表現。奇怪的是，我們一直無法利用shRNA由A549及CL1-5細胞中，篩選出具有穩定抑制PERP的細胞株，這情形暗示著PERP是肺癌細胞生存所必需的蛋白。由於有報導指出，高氧處理的細胞會過度表現PERP，這暗示細胞內因高氧產生的ROS可能會促進PERP的表現。而高代謝率的癌細胞也會產生較多的ROS，因此我們懷疑在肺癌細胞裡過度表現的PERP可能與ROS有關。為了探討PERP對肺癌細胞的影響，我們利用會引發細胞產生ROS的gallic acid處理細胞，觀察PERP的表現與細胞抗gallic acid的關係。結果顯示，利用si-RNA將內生性PERP的表現抑制後，A549及CL1-5細胞的侵犯能力被抑制，而細胞生長不受影響。但是，處理gallic acid之後，PREP被siRNA抑制的細胞生長速度較表現PERP的細胞慢。欲確定gallic acid抑制細胞生長與ROS的關係，我們以NAC (N-acetyl Cysteine)與gallic acid同時處理細胞，以降低gallic acid所產生的ROS。結果顯示，NAC可以解除gallic acid對不表現PERP細胞生長抑制的現象。接著，我們利用過度表現PERP的細胞，驗證PERP的表現可以抗gallic acid所產生的ROS。當CL1-0細胞過度表現wild type PERP及具有538 mutation的PERP時，細胞生長的速度與載體控制組相當。但處理gallic acid後，過度表現538 mtutation的PERP的細胞所形成之群落較載體控制組大，而過度表現wild type PERP的細胞，其群落大小則與載體控制組相當。由本研究，我們推測PERP蛋白在肺癌的細胞過度表現的功能有二；其一是與細胞的侵犯能力有關，其二是保護細胞免於ROS的傷害。因此，若能找出參與PERP保護細胞的機制，則可以發展抑制這個機制的方法，進而應用於治療肺癌上。

關鍵字

研究PERP

並列摘要

PERP (p53 apoptosis effect related to PMP-22) is a membrane protein with a tetra-span transmembrane domain. PERP is located at chromosome 6p24, a region implicated in the development of many kinds of cancers, such as ovarian, breast, cervical cancer and melanoma, as evident by frequent loss of heterozygosity (LOH) within this region. Due to the sequence homology, it is categorized to PMP-22 family. Previous studies has shown that PERP plays distinct biological roles in response to P53 or P63 regulation in different tissues. In MEFs, PERP is regulated by p53 and induces apoptosis. However, in epithelia cells, p63 regulates PERP that affects epithelial development and stratification. By IHC analysis on lung cancer tissue array, we observed that PERP was overexpressed in 83% of lung adenocarcinoma and 98% of squamous cell carcinoma. In lung cancer cell lines, A549, CL1-5, H1299 and H1355 expressed high level of PERP mRNA while CL1-0 and H661 has lower expression level. All cDNA clones derived from A549 and H1299 showed high frequency of multiple point mutations except one clone. A missense mutation at position 538 occurred in all mutated cDNA clones. To study the biological role of PERP in lung cancer cells, both wild-type and 538 mutated PERP was overexpressed in CL1-0 and CL1-5 while knockdown expression of PERP was performed in A549 and CL1-5 by siRNA. Intriguingly, none of the stable clones could be obtained when PERP was interfered by PERP-shRNA, indicating that PERP is essential for cancer cell survival. Since PERP has been reported to be up-regulated in cell grown under hyperoxia condition that may generate higher intracellular level of ROS, and since highly metabolic cancer cells have elevated ROS level, we hypothesized that over-expression of OPN may be related to the level of intracellular ROS. Therefore, we used a ROS generator, gallic acid, to treat lung cancer cells and observe the effect of PERP on gallic acid-induced cell growth inhibition. When PERP expression was interfered in A549 and CL1-5, the invasive ability of these two cell lines was inhibited while the growth of these cells was not changed compared with the siRNA control. However, when cells were treated with gallic acid, the growth rate of PERP knockdown cells was slower than the cells expressing PERP. To explore if the effect of gallic acid-induced cell growth inhibition in PERP knockdown cell could be mediated by ROS, NAC was applied during gallic acid treatment. The result showed that co-treatment of NAC has ability to diminish the gallic acid-induced growth inhibition in PERP knockdown cells. Furthermore, when CL1-0 cell over-expressing 538 mutated PERP, it was more resistant to gallic acid treatment as shown by forming larger colonies compared with that in vector control. Whereas, over-expression of wild-type PERP had colony formation ability the same as vector control. Our results suggested that over-expression of mutant PERP in lung cancer plays important roles in facilitating cell invasion and protecting cell from ROS damage. Discovery of the pathway involved in PERP-mediated cell protection from ROS would provide a potential strategy in cancer therapy.

並列關鍵字

PERP

參考文獻

Bae, Y. S., J. Y. Sung, et al. (2000). "Platelet-derived growth factor-induced H(2)O(2) production requires the activation of phosphatidylinositol 3-kinase." J Biol Chem 275(14): 10527-31.

Chambers, A. F., A. C. Groom, et al. (2002). "Dissemination and growth of cancer cells in metastatic sites." Nat Rev Cancer 2(8): 563-72.

Chiarugi, P. and T. Fiaschi (2007). "Redox signalling in anchorage-dependent cell growth." Cell Signal 19(4): 672-82.

Choi, M. H., I. K. Lee, et al. (2005). "Regulation of PDGF signalling and vascular remodelling by peroxiredoxin II." Nature 435(7040): 347-53.

Chu, Y. W., P. C. Yang, et al. (1997). "Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line." Am J Respir Cell Mol Biol 17(3): 353-60.

國際替代計量

研究PERP (p53 apoptosis effect related to PMP-22) 基因對人類肺癌細胞株生長的影響

全文下載

主題瀏覽