細胞的增生，分化或死亡是決定於細胞膜外的一些刺激像是有絲分裂素或是生長因子，去活化或抑制一些基因的表現。過去，本實驗室研究指出，利用yeast two hybrid技術，以P0蛋白為餌，於HeLa cDNA library篩選到GCIP (Grap2 and cyclin D interacting protein)。此外也利用RT-PCR的方式發現P0，一個真核生物60S核糖體蛋白，在乳癌病患檢體中有過度表現的現象，並且此現象不限於發生在是否有動情素接受器或Neu接受器的檢體中。在2002年Xiang等人提及，在已轉染人類端粒酵素的加速成長晶狀體上皮細胞中過度表現GCIP蛋白，此細胞株的生長速率會因此而減緩，這顯示GCIP在細胞的生長分化扮演壓抑的角色。

過去，本實驗室已經用哺乳類冷光酵素交互作用及免疫沉澱法證實P0 與GCIP間的確存在有交互作用，同時利用Mammalian two hybrid的方法來探討之間的交互作用，發現除了全長GCIP與P0具有交互作用外，其餘的GCIP片段並沒有交互作用。因此本實驗想要了解P0和GCIP的各片段區域(domain) 在in vitro是否存在有交互作用，所以採用GST pull down實驗，實驗結果指出，除了全長的GCIP可和P0交互作用外，去掉N端149 個氨基酸及去掉C端及Leucine zipper的truncated form GCIP也會與P0交互作用，因此，我們推測GCIP可能以Helix-loop-helix domain、acidic domain與P0結合。

此外，因GCIP與cyclin D1已證實有交互作用存在，但是對於有關cyclin D2、D3與GCIP的交互作用並未提及。在2001年Yu等人指出，在20 %的乳癌患者當中，有cyclin D1過度表現的現象，若將經由Ras或Neu等訊息傳遞所引起乳癌的下游蛋白Cyclin D1阻斷掉，則乳癌即不會發生，可見Cyclin D1的確在乳癌的增生中扮演作用標的的角色。因此若能確認GCIP與Cyclin D2、D3的關係，對於組織中特異性表現Cyclin D2、D3所導致的癌症，或許由P0的過度表現引起，提供一條可能的路徑。因此，利用免疫沉澱法及細胞內共同免疫沉澱法實驗結果發現，GCIP除了與Cyclin D1交互作用，亦與Cyclin D3有交互作用，並且相較於Cyclin D1，Cyclin D3與GCIP的結合能力是比較強的。

另外，挑選MCF7的P0 stable clone後，與送入未表現P0的質體以及不送任何質體的MCF7細胞相較，發現持續表現P0蛋白的細胞株生長速率較快。綜合以上結果，初步推測，在癌症中P0的過度表現可能藉由結合在GCIP的HLH domain 或Acidic domain傳送，影響下游Cyclin D1及Cyclin D3的細胞週期變化或細胞增生，至於調控的方式和機轉也是後續值得我們去探究的。

The regulation of gene expression of cell prolifiration, cell differentiation, and apoptosis are governed by extracellular stimuli like mitogens or growth factor. P0, a 60S ribosomal protein in eukaryotes, was a crucial component in a ribosomal stalk which plays an important role in polypeptide chain elongation during the translation step.Empolying quantitative RT -PCR (reverse transcription polymerase chain reaction) experiments, our preliminary studies revealed that overexpression of P0 was observed in breast cancer, which were with the expression of estrogen receptor-
positive or negative and with neu receptor-positive or negative.

In addition, using P0 as a bait, a novel gene GCIP (grap2 and Cyclin D interacting protein) was screened by yeast two hybrid method. Xiang et al. reported that GCIP overexpression in the pCl-hTERT-HLE (Human lens epithelial ) cells slows down the growth rate of the doubled-transfected cells. These suggested a potential repressor of GCIP in cell proliferation and differentiation. Although P0 bound to intact GCIP was confirmed in both yeast two hybrid assay and mammalian two hybrid assays, P0 interacted with which domain of GCIP had not been determined. In this study, in vitro GST pull down experiments was performed to investigate whether truncated forms of GCIP interact with P0 or not. The results revealed that P0 interact with truncated form of GCIP lacking leucine zipper domain and N-terminal region 149 amino acids, suggesting that GCIP interact with P0 through binding to HLH domain or acidic domain.

Although GCIP bound to Cyclin D1 in some cell line (Jurkat, COS-7) had been reported, whether GCIP could bind to Cyclin D2 and Cyclin D3 or not were unknown. The cyclin D1 gene was amplied in up to 20% of human breast cancer. Yu et al. reported that cyclin D1-deficient mice are resistant to breast cancers induced by the neu and ras oncogens. It was therefore propose that anti-Cyclin D1 therapy may be highly specific in treating human breast cancers with activated Neu-Ras pathways. If we determine the relationship between GCIP and Cyclin D2, D3, it may provide a possible link between P0 overexpression and Cyclin D2, D3 in specific cancers. In order to investigate wheather GCIP bound to Cyclin D2, D3 or not, Cyclin D1 and D3 were cloned from breast cDNA library, while Cyclin D2 was cloned from HeLa cDNA library, and TNT Quick Coupled transcription / Translation and immunoprecipitation (IP) were performed. Compared to the binding capability of Cyclin D1, that of Cyclin D3 is stronger.

Furthermore, MCF7 cells containing stably expressive P0 showed a significant increase in growth rate compared to MCF7 cells and MCF7 cells only containing pMH vector. In conclusion, our results indicate that P0 overexpression is involved in cancer signal transduction through binding to HLH domain or acidic domain of GCIP, then mediates downstream cyclin D1, D3. However, how GCIP influences cell cycle progression and cell proliferation of cyclin D1 and D3 remains to be further investigated.

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