利用小鼠腸道發炎模式系統來探討拓樸酶在氮化壓力所引起的DNA損傷扮演的角色

Abstract

潰瘍性結腸炎（ulcerative colitis）為一種腸道發炎疾病（inflammatory bowel diseases），其患者深受氮化壓力（nitrosylation stress）、腸道組織損傷及基因不穩定的影響，故較正常人更易形成大腸直腸癌；所以，我們想更進一步探討在發炎的情況下，氮化壓力的產生和癌症發生之間的交互作用與分子機制。實驗室先前的研究顯示氧化壓力具有引起第二型拓樸異構酶切割複合物產生的能力，進而導致DNA 斷裂、突變及細胞死亡；實驗室近期研究顯示一氧化氮可引起氮化壓力、產生第二型拓樸異構酶切割複合物（topoisomerase II cleavable complex, TOP2cc）及拓樸異構酶IIβ亞型之依賴性癌症；然而，第二型拓樸異構酶媒介之DNA損傷的活化是否在潰瘍性結腸炎所造成的組織損傷及進一步的癌症產生之間扮演不可或缺的角色是值得被進一步探討的。為了研究上述主題，我們利用化合物DSS引起之小鼠腸道發炎模式、免疫組織染色及藥物來探討在發炎的組織內，氮化壓力及DNA損傷的表現程度。我們觀察到在腸道發炎的小鼠內，第二型一氧化氮合成酶（nitric oxide synthase II, NOS2）大量表現，並且和巨噬細胞的表現趨勢一致，說明巨噬細胞的NOS2表現可能與DSS引起小鼠腸道發炎之病理進程有關；此外，我們也觀察到腸道發炎小鼠具有體重減輕及腸道縮短這些腸道發炎的特徵。我們利用第二型一氧化氮合成酶缺失的小鼠（NOS2-/-）探討氮化壓力是否在腸道發炎中扮演重要角色；我們觀察到體重減輕及腸道縮短在第二型一氧化氮合成酶缺失小鼠是較為緩解的；此外，我們也觀察到DNA損傷在第二型一氧化氮酶缺失小鼠也是減少的。以上結果顯示，對於腸道發炎所引起的病理現象及DNA損傷，一氧化氮是重要因子；且巨噬細胞在腸道發炎組織內為主要的一氧化氮來源。接著，我們進一步利用第二型拓樸異構酶針對性藥物來探討第二型拓樸異構酶是否可能在氮化壓力所引起的DNA損傷中扮演角色。我們發現在腸道發炎的小鼠內，第二型拓樸異構酶抑制型藥物（ICRF-193)具有減少DNA損傷的能力；以上結果顯示，第二型拓樸異構酶具有媒介發炎時氮化壓力所引起DNA損傷的能力。進一步研究第二型拓樸異構酶是否與發炎與發炎相關之癌症發生有關，我們利用腺病毒載體（adenoviral vector）表達Cre 重組酶（Cre-recombinase）於老鼠之大腸直腸細胞，將來將利用此方法來製造拓樸異構酶IIβ亞型缺失之TOP2βflox/flox 老鼠供研究拓樸異構酶IIβ亞型是否與發炎與發炎相關之癌症發生有關。

Patients with ulcerative colitis (UC), an inflammatory bowel disease suffered from nitrosylation stress, colon tissue damage and genomic instability, are prone to tumor formation. Here, we have investigated the contributions of inflammation-associated nitrosylation stress to UC pathogenesis and its underlying mechanisms. Previously, we have reported that oxidative stress induces topoisomerase II cleavable complex (TOP2cc) formation and subsequently leading to DNA breakage, mutagenesis and cell death. Moreover, we have also recently shown that nitric oxide (NO) induces nitrosylation stress, TOP2cc formation and TOP2β-dependent tumor formation. However, it remains to be determined whether NO and TOP2-mediated DNA damage plays an important role in UC-associated tissue damage and cancer development. To address this, immunohistochemistry (IHC) approach was first used to examine the expression of inducible nitric oxide synthase (NOS2, indicative of NO production) and γ-H2AX (indicative of DNA breakage) in colon of mice suffered from dextran sodium sulfate (DSS)-induced UC. Consistent with previous studies, we observed an increase in NOS2 expression and macrophage infiltration in the colon tissue samples from DSS-treated mice compared to control mice. In addition, we found that DSS induced concentration-dependent loss in body weight and shortening of colon length. Consistent with the notion that NO plays an important role in the pathogenesis of DSS-induced UC, all the above DSS-induced phenotypes are much relieved in the NOS2 knockout (NOS2-/-) mice. Furthermore, our results revealed for the first time that γ-H2AX signal is much higher in WT mice than NOS2-/- mice suggesting that nitrosylation stress which is responsible for DSS-activated DNA damage response and subsequent tissue damage. Further usage of TOP2-targeting drugs, VP-16 and ICRF-193, are used to determine the role of TOP2 in nitrosylation stress-induced DNA damage. Mice treated with ICRF-193 showed reduced DNA damage compared with DMSO-treated mice under DSS treatment with any significant influences on body-weight loss, colon-length shortening and inflammatory score. Together, our results suggest that TOP2 mediates nitrosylation stress-induced DNA damage. Future investigation by using the tissue-specific knockout of TOP2βflox/flox mice were needed to determine the role of TOP2β in nitrosylation stress as well as associated mutagenic and carcinogenic activity.