2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660801摘要：此申請案中，我們計劃使用生化、遺傳、細胞的實驗方法及動物模式系統來研究兩種第二型拓撲酶(TOP2α與2β)，在氧化/氮化逆境下相關的基因突變和細胞凋亡中所扮演的角色。在我們與其它的研究都指出，藉由DNA 拓撲酶所造成的DNA 斷裂，是有效的抗腫瘤治療策略之一。然而，TOP2 造成的DNA 損傷也提高了基因突變的機率，可能導致繼發性惡性腫瘤的發生。生理逆境（如氧化反應和酸中毒）、TOP2 的藥物（VP - 16，臨床藥物），可以有效地活化TOP2 造成DNA 斷裂，導致後續的基因突變。且我們在兩階段的皮膚癌模型研究結果中，發現TOP2 造成DNA 斷裂與腫瘤促進劑TPA 一樣可以促進腫瘤發生。同時，研究亦指出組織的慢性發炎反應中的自由基（ROS / RNOS）可能會增加罹癌的風險。因此，我們推測發炎過程中產生的自由基可能活化TOP2 造成DNA斷裂，使得染色體DNA 不穩定（CIN），而加速癌症的發展。我們的初步結果中發現，各種癌細胞株在巨噬細胞或化學產生的自由基(GSNO)之環境下，會活化TOP2 而造成DNA 斷裂；同時TOP2 缺失的細胞株在DNA 斷裂和細胞死亡上呈現出更高的耐受性。研究目標如下：1. 確認RNOS 所引起的DNA 斷裂有TOP2 的參與。2. 研究TOP2 在氧化/氮化逆境下所引發的基因突變與細胞死亡中的角色。探討TOP2 是否參與發炎、癌前病變及腫瘤DNA 損傷的過程。<br> Abstract: The long-term objective of our lab is to understand the molecular mechanism(s) as well asphysiological function(s) of multiple DNA topoisomerases. In this current application, acombination of biochemical, genetic and cellular approaches will be utilized to study theproposed roles of the two topoisomerase II isoforms, TOP2α and 2β, in oxidative/nitosylatedstress and its associated mutagenesis, cell death and carcinogenesis. Numerous studies haveproven the induction of topoisomerase-mediated DNA break as an efficient anti-cancertherapeutic strategy. Nevertheless, TOP2-mediated DNA damage is also highly mutagenicwhich has been suggested to be involved in the generation of secondary malignancies. Wehave previously reported that physiological stresses (e.g. oxidative stress and acidosis), likeTOP2-targeting etoposide, could activate TOP2-mediated DNA breakage and leading tosubsequent mutagenesis. Strikingly, our results also showed that TOP2-mediated DNAbreakage exhibit a tumor-promoting activity like that of TPA in the two-stage skincarcinogenesis model. In addition, an increased cancer risk has been reported to occur intissues undergoing chronic inflammation, in which the generation of free radical species [e.g.reactive oxygen/nitrogen species (RNOS)] and associated mutagenic events has beensuggested to be responsible for the onset of carcinogenesis. We therefore hypothesized thatthe oxidative/nitrosylated stress activates TOP2-mediated DNA breakage during chronicinflammation, leading to activation of DNA damage responses, chromosome instability and apotential acceleration of subsequent carcinogenesis. In the preliminary results, we have indeedobserved that the activation of TOP2-mediated DNA breakage on chromosomes in variouscancer cell lines during chemical- and macrophage-associated oxidative/nitrosylated stress.Consistent with the involvement of TOP2, TOP2-deficiennt cell lines are more resistant toH2O2- and nitric oxide (NO)-induced chromosome DNA fragmentation and cell killing. Inaddition, cellular exposure to a NO-releaser GSNO increases mutation frequency. Consideringthe potential importance of oxidative/nitrosylated stress in mutagenesis and carcinogenesis,investigation of TOP2 should be warranted. Three specific aims are listed as the following:1. To mechanistically dissect the involvement of TOP2 in the RNOS-induced DNAbreakage: First, the in vitro cleavage with recombinant enzymes will be used to examinethe ability of NO in the induction of TOP2-mediated DNA breaks. We will theninvestigate the novel mechanism for TOP2-mediate DNA cleavage induced by RNOSwith the primary focus on the potential contribution of thiol-modification on TOP2.2. To determine the differential role of TOP2 isozymes in mutagenesis, carcinogenesisand cell death induced by oxidative/nitrosylated stress: The potential contribution(s) ofTOP2α and 2β in inflammation-associated mutagenesis, cell killing as well as skin tumordevelopment will be examined and revealed using RNAi technology, conditional knockoutmouse and the two-stage skin carcinogenesis models3. To explore whether TOP2 is involved in the activation of DNA damage responses ininflamed tissues, precancerous lesions and during tumorigenesis: Key parameters,such as DNA damage molecules, at different tumorigenic stages the two-stage skincarcinogenesis models using various TOP2-targeting agents or TPA will be determined.Particularly, we highly anticipate that the extent of oxidative/nitrosylated stress and thelevel of DNA damage responses should be positively correlated.