研究KLF4 調控之端粒酶活化作用及尋找端粒複製的抑制物

Abstract

端粒酶為保護線性染色體動態避免基因組不穩定極端點融合的DNA-蛋白質複合體。大部分的端粒DNA由端粒酶合成，幹細胞則定義為在有絲分裂及分化時期有能力自我更新至不同特殊細胞種類的細胞。 KLF 家族為參與調控不同細胞生理現象的轉錄因子，例如發育、分化、增值以及細胞凋亡。KLF4 為此家族成員之一，藉由與不同的蛋白交互作用執行轉錄活化或抑制的功能。我們利用質譜儀發現KLF4上有潛在性被MAPK所磷酸化的位點，此外，我們也鑑定出一些與KLF4有交互作用的蛋白質。我們利用不同的細胞株去觀察特定的交互作用及磷酸化現象。由於在癌細胞及幹細胞中p21 及hTERT 為KLF4主要的調控目標，我們的目標為闡明KLF4在癌細胞中調控p21及幹細胞中端粒酶調控的分子機制及生物意義。 癌細胞可藉由端粒酶或DNA重組的方式延長端粒，在本研究中，我們探討端癌細胞粒酶重組的機制。我們發現在酵母菌及癌細胞中DNA拓樸酶二與三為端粒重組所必須，拓墣酶二解重組DNA高正向超螺旋結構而拓墣酶三解重組DNA高負向超螺旋結構，拓樸酶二的抑制劑Mitoxantrone，可干擾端粒重組並導致端粒縮短並影響ALT細胞的特性。 目前並沒有藥物可以治療ALT 癌症，所以我們正在找尋拓墣酶二抑制劑可以抑制ALT路徑，在之前的證據指出，於ALT細胞中剔除拓墣酶三，可重新啟動端粒酶活化路徑，我們也希望能找到治療ALT及其衍生性癌症的方法。

Telomeres are dynamic DNA-protein complexes that protect the ends of linear chromosomes from genome instability and end fusion. Most telomeric DNA is synthesized by the enzyme telomerase. Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. The KLF families are transcriptional factors that regulate a diverse array of cellular processes, including development, differentiation, proliferation and apoptosis. KLF4, a member of this family, functions as a transcriptional activator or repressor depending on the interaction partner and the context of the binding sites. We have identified a potential MAPK phosphorylation site on KLF4 by mass spectrometry. Additionally, multiple KLF4 interacting proteins were identified. All these modification and interactions may define the specificity and refine its roles in different cells. Since the major targets of KLF4 are p21 in differentiated somatic cells and telomerase in undifferentiated stem cells, our goal is to elucidate the molecular mechanism and biological contribution of KLF4-mediated p21 repression in cancer cells and telomerase activation in stem cells. Cancer cells can elongate telomere either through telomerase reactivation or through an alternative recombination pathway for telomere lengthening (ALT). In this study, we investigate telomere recombination in mammalian. We have shown that topoisomerase 2 and 3 are all required for the ALT in yeast cells. Topoisomerase 2 resolves hyperpositively supercoiled DNA before recombination forks and topoisomerase 3 resolves hypernegatively supercoiled DNA after recombination forks. Topoisomerase 2 and 3 are not required for telomere recombination when supercoiled DNA is not formed. Adding topoisomerase 2 poison, mitoxantrone, in medium could also conferred telomere shortening and alter many phenotypes in ALT cells. So far there is no drug developed to inhibit ALT, so we are looking forward to find TOP2 poisons or inhibitors can inhibit both Type II recombination and ALT pathway. In previous data showed that knockdown of TOP3 in ALT cells could reinitiate telomerase activity for cell survive, we are also wish to find a treatment combine both telomerase inhibition and ALT blockage for complete therapy in ALT cancer.