探討人類酪胺酸DNA磷脂酶在處理拓樸異構酶可切性複合體上所扮演的角色

Study on Roles of Human Tyrosyl-DNA Phosphodiesterase 1 in processing of Topoisomerase Cleavable Complexes

彭安林(An-Lin Peng)

指導教授 ： 李財坤

國立臺灣大學/醫學院/微生物學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.02576

摘要

引發拓樸異構酶可切性複合體(TOPcc)被認為是有效的抗微生物以及抗癌策略，因其可以造成有蛋白質連結在3端或5端DNA斷點上的特殊DNA傷害。人類酪胺酸DNA磷脂酶(human tyrosyl-DNA phosphodiesterase 1, hTDP1)藉由其水解3’磷酸二脂鍵的能力而參與在處理以及修復TOP1cc所造成的DNA傷害過程中。然而，hTDP1在處理TOP2cc中是否扮演腳色，以及和TOPcc的其他細胞處理途徑(複製起始的處理：RIP，和轉錄起始的處理：TIP)之間的關係，仍不清楚。因此，本論文利用藥物調控、RNA干擾技術，和強制蛋白質表現等策略來探討hTDP1對TOP2cc引發的DNA傷害及反應之影響。
我們分別在HCT116細胞株和BT-474細胞中降低hTDP1的蛋白質表現；和控制組細胞比較，發現hTDP1缺乏的細胞對於拓樸異構酶II藥物所造成的DNA斷點較多，且對於藥物的敏感性也顯著增加；相反地，在293細胞以及hTDP1缺乏的細胞中強制其蛋白質表現則可有效抵制上述表徵。此外，clonogenic assay的結果也支持著hTDP1對於TOP2cc引發之細胞死亡有著保護性的腳色。而hTDP1的缺乏顯著地增加了細胞對於TOP1和TOP2藥物引起之NHEJ事件，更加支持hTDP1可能在細胞處理TOP2cc的過程中佔有一席之地。
接下來，為了探討hTDP1和RIP/TIP的關係，我們研究兩者對TOP1cc的差異。首先，我們發現缺乏hTDP1並不影響TOP1cc的降解，表示hTDP1可能作用於蛋白體降解(proteasome degradation)之後，或是作用於和TIP路徑平行的RIP路徑。根據實驗室先前對於不同處理途徑所引發特定的TOP1藥物造成之DNA傷害反應，相較於控制組細胞，我們發現hTDP1缺乏的細胞處理TOP1藥物後能引發較高程度的Chk2和p53活化(TIP引發的傷害訊息分子)，而RIP引發的傷害訊息分子，DNA-PK和RPA的活化則沒有顯著差異：使用藥物分別抑制複製和轉錄也得到一致的結論。因此，hTDP1可能經由TIP路徑活化TOP1藥物所引發下游的DNA傷害反應。總括而論，本研究可提供合併療法在機制上的解釋，並對於以TOP作為抗癌標的的治療方針的效益提升貢獻了可能的幫助。

關鍵字

人類酪胺酸 ； DNA磷脂酶拓樸異構酶可切性複合體

並列摘要

Formation of topoisomerase cleavable complex (TOPcc), in an unique form of protein-linked 3’- or 5’-DNA breakage, represents as an effective antibiotic and anti-cancer strategy. Human tyrosyl-DNA phosphodiesterase1 (hTDP1) has been implied in TOP1cc processing and repair through the activity of hydrolyzing 3’-phosphotyrosyl bond between TOP1 and DNA. However, the role of hTDP1 in TOP2cc processing and the relationship among hTDP1, TIP and RIP pathways remain unclear. Toward this aim, a combination of pharmacological modulation, RNAi technology and forced expression approach was utilized to investigate the impact of hTDP1 on TOP2cc-mediated DNA breakage and damage responses (DDR).
Two pairs of hTDP1-knockdown and control cells had been successfully established in HCT116 cells and BT-474 cells. Our results showed hTDP1-deficient cells exhibited a higher extent of DNA breakage and hypersensitivity to TOP2-targeting drugs. While forced expression of hTDP1 in 293 cells and hTDP1-deficient cells could reduce TOP2-mediated DNA damage and counteract these hTDP1-deficient phenotypes. The clonogenic assay additionally supported the protective role of hTDP1 in TOP2cc-mediated cell killing. Moreover, the increased rate of CPT- and VP16-induced NHEJ-mediated plasmid integration in hTDP1-deficient cells also consistent with the potential involvement of hTDP1 in the processing of TOP2cc.
In regarding to the relationship between hTDP1 and cellular processing pathways of TOP1cc, we first observed that CPT-induced downregulation of TOP1cc was not affected in hTDP1-deficient cells. Two possible explanations were offered: (i) hTDP1 could be involved in the downstream of proteasome degradation. (ii) hTDP1 could be involved in RIP, which is parallel with proteasome degradation. Based on our previous study on distinct activation of CPT-induced DNA damage responses (DDR) via two processing pathways, we further observed that phosphorylations of Chk2 and p53 (TIP-related signals) rather than DNA-PK, RPA (RIP-related signals) were increased upon CPT treatment in hTDP1-deficient cells. Similar conclusion was also obtained from pharmacological modulation studies. Therefore, our data suggest a potential contribution of hTDP1 to CPT-induced DDR through TIP pathway. In sum, our studies might therefore provide mechanistic explanations for these combination effects and further helps for the development of TOP-based therapeutic intervention.

並列關鍵字

hTDP1 ； topoisomerase ； cleavable complex

參考文獻

Antony, S., Marchand, C., Stephen, A.G., Thibaut, L., Agama, K.K., Fisher, R.J., and Pommier, Y. (2007). Novel high-throughput electrochemiluminescent assay for identification of human tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors and characterization of furamidine (NSC 305831) as an inhibitor of Tdp1. Nucleic Acids Res 35, 4474-4484.

Aplan, P.D. (2006). Chromosomal translocations involving the MLL gene: molecular mechanisms. DNA Repair (Amst) 5, 1265-1272.

Austin, C.A., and Marsh, K.L. (1998). Eukaryotic DNA topoisomerase II beta. Bioessays 20, 215-226.

Austin, C.A., Sng, J.H., Patel, S., and Fisher, L.M. (1993). Novel HeLa topoisomerase II is the II beta isoform: complete coding sequence and homology with other type II topoisomerases. Biochim Biophys Acta 1172, 283-291.

Baguley, B.C., and Ferguson, L.R. (1998). Mutagenic properties of topoisomerase-targeted drugs. Biochim Biophys Acta 1400, 213-222.