摘要：計劃背景正常細胞之各種生理功能之協調需要非常複雜的調控機制。若是調控失靈，就可能引發惡性腫瘤之生成。目前已知，基因突變、過度之基因甲基化、與不正常之訊息傳遞均與癌症之生成有關。其中，NF-kB、b-catenin、c-MYC、與 p53 等轉譯因子在癌細胞中之訊息傳遞異常已有較為廣泛之研究。雖然近年來，這些異常在血液腫瘤中已有一些報導，仍有很多問題需要解答。首先，傳統之檢驗轉譯因子活性的方法包括利用螢火蟲螢光蛋白為受質、細胞核內蛋白質染色或是電泳移動轉變的方法。但是這些方法的缺點包括無法分析自病人抽取之血液中的特定細胞族群、非定量、非直接反應出轉譯因子之活性、緩慢及費力。第二，雖然有一些零星文獻顯示這些轉譯因子之間有互相影響，但是在血液腫瘤中，這類的關聯性仍然晦暗不明，有待進一步釐清。第三，這些轉譯因子之異常活性與各種基因突變、表基因變異、臨床表現均有關聯。文獻顯示NPM1 突變常合併NF-kB 活性降低；而FLT3/ITD 常合併b-catenin 過度活化；並且一些b-catenin 之抑制因子之基因過度甲基化常導致b-catenin 之過度活化。這些彼此之間的關聯性將需要更深入之探討。。第四，不同的骨髓性腫瘤其訊息傳遞之異常是否有所不同？第五，當這些骨髓性腫瘤被好好控制時或是疾病進展時，其訊息傳遞之異常會怎麼樣變化？本計劃之目的在於解決這些問題。材料與方法我們將採用一種短半生期的綠色螢光接在各種轉譯因子的結合核苷酸序列之後，以反映該轉譯因子之活性。而且同時置入一個本來就有高表達性之紅色螢光載體，以作為矯正因素。再配合anti-CD45 抗體與Log SSC，此系統可以使用於流式細胞儀，我們將可輕易分析各種特定之血液細胞族群，特別是含有各式細胞之骨髓化生不良症狀群。各種訊息傳遞之間均有交互作用，而且其異常與基因突變也可能有關聯。而一些基因之過度甲基化與異常之訊息傳遞也有密不可分的關係。我們希望藉由這些廣泛性之研究來釐清更多彼此之間的交互作用，並進一步探討與臨床表現之間的關聯性。預期結果我們相信本研究計劃將可提供一個特有的平臺以分析人類血液腫瘤中訊息傳遞之異常，並且整合分析訊息傳遞異常、基因與表基因變異與臨床表現的關聯性。我們也將有機會檢示不同之骨髓性腫瘤之間的生物性的差異。最重要者，我們可以分析這些血液病在惡化時或是經妥善治療後，訊息傳遞之活性的變化。這一系列之資料將有助於我們了解各種骨髓性腫瘤之致病機轉與臨床意義。
Abstract: BackgroundMalignant transformation of cells results from disruption of the delicate balance amongcomplex biological processes. Genetic mutation, aberrant methylation, and abnormalactivation of signal transduction pathways are well known etiologies of cancers. NF-kB,b-catenin, c-MYC, and p53 are transcription factors, whose aberrancies have been extensivelystudied in solid tumors. Although inappropriate control of these pathways is also present inblood diseases, many important questions remain to be answered. First, ideal methodologyfor comprehensive characterization of these pathways in human blood cells has not beenestablished. Traditional methods of measuring transcription factor activities such asluciferase reporter assay, immunohistochemical (IHC) staining of the nuclear proteins, orelectrophoretic mobility shift assay (EMSA) are either unable to analyze discrete cellpopulation in human blood cells, non-quantitative, not directly reflecting the functional levels,low throughput, or tedious. Secondly, the interaction among these pathways in humanmyeloid malignancies remains elusive, although a few studies have indicated mutualinteraction among NF-kB, b-catenin, c-MYC, and p53 pathways. Thirdly, the relationshipamong the abnormalities of these signal transduction pathways, clinical parameters, andgenetic/epigenetic alteration has not been explored. Current literature suggests that geneticmutations such as NPM1 and FLT3/ITD are associated with decreased NF-kB and enhancedb-catenin pathway activation, respectively, and that aberrant methylation in many antagonistsof b-catenin activation is present in malignant cells. Fourthly, the difference of signaltransduction activities among various myeloid malignancies (AML, MDS, cMPD) has notbeen investigated. Fifthly, the change of the aberrant transcription factor activities whendiseases are well controlled or in progression is not known. The main purpose of this projectis to solve these problems.Materials and MethodsWe will develop a flow cytometry-based reporter system, in which protein-binding DNAsequences are fused with short half-life version of green fluorescence protein (dscGFP),whose intensity represents the activities of the transcription factors under interrogation. Theinternal control of transfection efficiency will be reflected by a constitutively expressed red2protein, dsRed, driven by CMV promoter. By co-transfection of dsRed and dscGFP reporterplasmids into the primary human cells, followed by staining with anti-CD45-PC5, we are ableto analyze disparate cell populations by gating function in flow cytometry. Different cellssuch as blasts, monocytes, lymphocytes, and neutrophils can be discerned by combination ofanti-CD45-PC5 and log SSC. The FL1 intensity of GFP represents the transcription factoractivity. The loading and transfection efficiency can then be corrected by dsRed intensity inanother channel FL2. The intrinsic difficulty in transfection of suspension primary cells willbe solved by a new platform of electroporation. We believe this novel reporter assay wouldenable us to better analyze hematological diseases, such as MDS. Secondly, by parallelmeasurement of these transcription factor activities, we would be ready to analyze theinteraction of these discrete pathways. Thirdly, the activities of these signal transductionpathways can be further analyzed based on the genetic/epigenetic alterations, clinicalparameters such as cell count, age, gender, and survival. Fourthly, by comparing theabnormalities of these signal transduction pathways in AML, MDS, and cMPD, we can gaininsight of the biological differences among these diseases. Finally, we will analyzesequential patient samples to learn how signal transduction abnormalities change whendiseases progress or treated.Expected resultsWe believe that our current project will provide a unique platform for functional analysis ofsignal transduction in primary human hematopoietic cells, and will integrate the aberranciesin signal transduction, biological phenotypes, and clinical characteristics of patients withblood malignancies. We will get an opportunity to examine the biological difference amongdifferent myeloid malignancies. Most importantly, by following up sequential samples ofsame patients, we can investigate the change of signal transduction abnormalities whendiseases are controlled or progress, thus culminating in understanding their complexpathogenesis and clinical significance.