人類類胰島素生長因子結合蛋白-4對鼻咽癌細胞株生長速率的影響

Abstract

鼻咽癌是國人常見的癌症疾病。為了了解鼻咽癌的成因，我們進行鼻咽癌細胞與一般上皮細胞各種基因表現量的比較分析，發現在鼻咽癌細胞中類胰島素生長因子結合蛋白-4 (Insulin-like Growth Factor Binding Protein-4, IGFBP-4) 表現量的降低。類胰島素生長因子 (Insulin-like Growth Factor, IGF) 具有促進個體生長、代謝、細胞增長、分化等多種功能，經由IGF的受器 (IGF Receptor, IGF-R) 與IGFBP交互調節其功能。由於在乳癌、大腸癌、骨癌與攝護腺癌等例子中發現，IGFBP的不正常表現，有可能與這些癌症的形成相關。本實驗之目的即在探討是否IGFBP-4的表現與鼻咽癌的成因相關。為了得到完整的IGFBP-4 cDNA序列，首先我們篩選了人類大腦、肌肉與肝臟的cDNA基因庫，得到一個約1.6 kb的IGFBP-4 cDNA選殖株，命名為pIB403；並且剪接至表現載體pET30C，命名為pIB406。由於pIB403未包含完整的IGF結合序列，我們抽取了肝癌細胞株HepG2的總體RNA，以反轉錄-聚合酶連鎖反應的方式選殖IGFBP-4的cDNA的轉譯區的序列，得到包括完整訊息片段的pIB404，並且剪接到表現載體pGEX-5X與pET30送入大腸桿菌都能大量表現。這些選殖株以低溫誘導與山梨醣醇-甜菜鹼培養的方式表現，仍無法得到可溶性的蛋白質；而無法以外加至培養液的方式，來比較鼻咽癌細胞株生長情況的變化。同時，我們將Dr. Mohan所贈與完整的IGFBP-4 cDNA株剪接至載體pcDNA3分別轉染鼻咽癌細胞株NPC-TW039與NPC-BM1。由於NPC-TW039不容易進行轉染，並未觀察到IGFBP-4大量表現對NPC-TW039的影響；而在NPC-BM1細胞株中，大量表現IGFBP-4的轉染細胞株，生長速率明顯較對照組 (未轉染或僅轉染pcDNA3的細胞) 慢，顯示在鼻咽癌細胞株中，IGFBP-4表現量增加的確會對鼻咽癌細胞株的生長造成抑制的作用。

Nasopharyngeal carcinoma (NPC) is a common cancer disease in Taiwan. PCR-subtraction technique had been employed to investigate the differential gene expression between normal epithelium cells and nasopharyngeal carcinoma cells. Lower expression level of insulin-like growth factor binding protein-4 (IGFBP-4) in NPC cells has been noted. Insulin-like growth factors (IGFs) play an important role in promoting cell differentiation and proliferation. The functions of IGFs are modulated by IGFBP and IGF receptors. The abnormal expression of IGFBPs has been reported in many cancers including bone cancer, breast cancer, colon cancer and prostate cancer, suggesting a likely role in tumor formation. In order to study the relationship between IGFBP-4 expression and NPC formation, we have initially tried to screen human cDNA libraries for a full length cDNA clone using an EST clone as probe. An incomplete clone pIB403 which contains 1.6 kb DNA with partial IGF binding domain was isolated. The 500 bp IGFBP-4 coding sequence from pIB403 was then subcloned to expression vector pET30C and named pIB406. In order to obtain a clone with intact IGF binding domain, we have also isolated a clone pIB404 via RT-PCR using the RNA extracted from HepG2 as template. The 800 bp coding sequence from pIB404 was also subcloned into expression vectors pGEX-5X and pET30, respectively. We were able to overexpress these recombinant clones in Escherichia coli. However, neither incubation under lower temperature (30℃) nor growth bacteria under sorbital-betaine condition could help to obtain soluble IGFBP-4 protein for further study. We have also tried to transfect two NPC cell lines, NPC-TW039 and NPC-BM1 with an IGFBP-4 full length cDNA sequence subcloned in pcDNA3. In NPC-TW039 cells, the effect of IGFBP-4 overexpression was not detected possibly due to low transfection efficiency. Proliferation of the IGFBP-4 transfected NPC-BM1 cells appeared to decrease, suggesting an inhibitory effect of IGFBP-4 on growth of the NPC-BM1 cells.