人類細胞中的hRad9蛋白(Human homologue of Schizosaccharo- myces pombe Rad9)是一個在演化上具有高度保留性的蛋白。hRad9由391個胺基酸所組成，並具有三個區域，分別為N端的兩個PCNA-like區域以及C端區域。文獻指出hRad9在細胞內扮演著許多重要的角色，包括調節細胞週期的進行43、執行DNA的修復32、誘導細胞凋亡18的產生以及調控部分基因的轉錄53。此外也陸續有研究指出hRad9的其他功能，例如維持整個基因體的完整性23，促進核糖核甘酸(ribonucleotide)的生合成以及它具有3’端到5’端核酸外切酵素(exonuclease)的活性。基於Rad9所具有的這些功能，便有文獻指出Rad9在細胞內所扮演的角色與腫瘤抑制因子(tumor suppressor)p53極為相似18，然而之前許多研究已明確指出細胞內p53的表現主要是受Mdm2(murine double minute 2)這個蛋白所調控。所以我們實驗室便想要探討Rad9與Mdm2這個負向調控因子是否存在著某種關係。在實驗室之前的研究已利用共同免疫沉澱以及免疫螢光染色等實驗發現Rad9與Mdm2有交互作用的現象產生。所以接下來我們想要進一步的探討Rad9是否會影響Mdm2及p53蛋白的表現或穩定(stability)。首先我們利用核醣核酸干擾技術抑制細胞內Rad9蛋白的表現，從實驗結果我們發現當Rad9的表現量降低時，Mdm2的表現是呈現上升的趨勢;相對的，p53的表現則有下降的現象產生。此外我們也進一步讓細胞能過度表現Rad9蛋白，發現當Rad9的表現量增加時，Mdm2的表現是呈現下降的趨勢，此時p53的表現則呈現上升的現象。除了觀察Rad9是否會影響Mdm2及p53的蛋白表現，我們也探討Rad9是否會影響Mdm2及p53 mRNA，實驗結果顯示Rad9並不會影響這兩個蛋白的轉錄。由此可知Rad9影響Mdm2及p53的表現主要是在轉譯(translation)或轉譯後修飾作用(post-translation modification)的層次。接著我們更利用cycloheximide這個轉譯作用的抑制劑探討Rad9對Mdm2及p53半衰期(half-life)之影響，從實驗結果我們發現，當Rad9的表現降低時，Mdm2的半衰期相對於控制組則有延長的現象產生；相反的，p53的半衰期則呈縮短的情形。此外我們也利用實驗發現Rad9會影響Mdm2及p53的泛素化(ubiquitynation)。當Rad9的表現降低時，Mdm2的泛素化是呈現趨緩的現象;相對的，p53的泛素化則有增加的情形產生。然而在之前的文獻已明確指出紫外光照射所造成的細胞損傷，是促使p53活化的主要原因之一。所以我們便想探討細胞在經過紫外光照射而造成DNA損傷過程中，Rad9是否也會影響Mdm2和p53的表現。在我們的實驗結果可以觀察到當細胞受到紫外光照射後，Rad9會影響Mdm2的降解(degradation)及p53的累積(accumulation)時間。綜合以上的實驗結果，我們得知Rad9對於細胞內Mdm2及p53的表現扮演著一個重要的角色，這也意味著我們似乎發現另一個調控Mdm2-p53路徑的新機制。

Human RAD9 was originally identified as a structural homologue of yeast Schizosaccharomyces pombe rad9 and is capable of partially complementing the radiosensitivity. The eukaryotic Rad9 genes are evolutionarily conserved. The encoded mammalian proteins participate in promoting resistance to DNA damage, cell cycle checkpoint control, DNA repair, and apoptosis. Other functions include a role in maintaining genomic integrity, and the transactivation of multiple target genes. Previous studies indicate that RAD9 functions much like p53, which controls multiple downstream pathways including cell-cycle regulation, apoptosis and maintain genomic integrity. The tumour suppressor p53 is kept labile under normal conditions. In unstressed cell, the anti-proliferative effects of p53 are restrained by mouse double minutes 2 (Mdm2), an ubiquitin ligase that promotes p53 ubiquitination and degradation. Mdm2 also mediates its own degradation through auto-ubiquitination. hRAD9 and p53 have important roles in cell cycle checkpoint control and apoptosis, suggesting that these two proteins might function coordinately. In our previously study, we found that Rad9 could interact with Mdm2. In this study, we provide evidences that Rad9 could influence Mdm2 stability. Knocking down Rad9 expression by siRNA led to an increase in Mdm2 levels and a decrease of p53 levels in several of human cell lines. Conversely, overexpression of Rad9 led to a decrease in the levels of Mdm2 and an increase in p53 levels. The effect of Rad9 on Mdm2 and p53 steady-state levels is not due to changes in their transcription because Rad9 does not alter the Mdm2 and p53 mRNAs in human A549 cells. Further, we found that Rad9 could modulate the half-life of Mdm2. Downregulation of Rad9 expression prolonged the half-life of Mdm2 in A549 cells and HEk293T cells. In addition, we also found that Rad9 could influence the ubiquitination of Mdm2 and p53. Knocking down Rad9 decreased in Mdm2 ubiquitination and increased in p53 ubiquitination. UV damage is one of the major stresses that provoked p53 action. In this study, we found that Rad9 could influence the endogenous protein levels of Mdm2 and p53, when cells were damage by UV. The siRNA-mediated knockdown of Rad9 resulted in marked delay in the accumulation of p53 and prolong in the accumulation of Mdm2, when cells were irradiated by UV. These results indicate that Rad9 seems to have a critical role in the control of the cellular levels of Mdm2 and p53 in response to DNA damage. Here, we have provided an additional mechanism for the control of Mdm2 function and a model for the involvement of Rad9 in the control of p53-Mdm2 pathway.

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