Abstract
The data collected enclosed in this thesis are roughly divided into fivesections. At first, we described the method of isolating porcine spleentopoisomerase II, and secondary, the characterization of such preparedtopoisomerase II. In the third section, we described a new method andbased on Sanger dideoxy DNA sequencing method and topoisomerase IIreaction mechanism to elucidate the DNA recognition and cutting sites oftopoisomerase II. Then a new topoisomerase II inhibitor, withangulatin a,with the low dossage effect of enhancing the DNA-topoisomerase IIcleavable complwx and with high dossage effect of blocking theinteractions between DNA and topoisomerase II is described in the fouthsection. At last, several kinds of drugs of various DNA and topoisomeraseII binding activity were used by way of inhibiting the formation ofcleavable complex enhancing by mANSA or VM-26 to deduce the fundamentalcharacters of the anti-cancer drugs which can induce the topoisomerase IImediated DNA damage.(1) There are two types of topoisomerase II present in porcine spleen. Twofotms of DNA topoisomerase II with relative molecular weight of 160 KDaand 150 KDa on denatured SDS PAGE have isolated from porcinespleen.Although the in situ peptide maps of these two polypeptides aredifferent from each other, the peptide map of them labeled with [32p] byprotein Kinase C or myelin basic protein Kinase are just the same. Boththe two antiserums each prepared from the two polypeptides can bind humabntopoisomerase II protein, too.(2) Characterization of porcine spleen topo-isomerase II.THE DNA dependent ATPase activity of our prepared topoisomerase II dependson the concentration of ATP itself. When the concentration of ATP issmaller than 0.2 mM, the Km valure of it is 0.01 mM. And when the ATPconcentration of ATP is larger than 0.2 mM,both the value of Kmand Vmincrease. Anti-cancer drugs, VM-26 and wothangulatin A, and calcium ioncan uncouple ATP hydrolysis with the activity of DNA strand passage by thetopoisomerase II.(3) The topoisomerase II DNA cutting sites.A novel set of specific DNA cutting sites atound plasmid Col El ori andthe 5' upstream region of human hsp70 gene by porcine topoisomerase IIwere found, and these recognition sites are changed cinsiderably by thepresence of ATP and histone core.The base adenine was detectded at 3'-OHend if the porcine topoisomerase II cutting site for 10 cases aming the 12cutting sites screened with the following consensus sequence: A/T -C N PyPu N Pu A Py A^PuN -A -C N N N G/C Pu N we determined.(4) Withangulatin A--a new topoisomerase II inhibitor enhancingtopoisomerase II mediated DNA damage.Many topoisomerase II inhibitors were screened out from isolatedcomponents of chinese herbs. Among them, withangulatin Aisolated fromPhysalis angulata L was found to act on topoisomerase II to inducetopoisomerase II mediated DNA damage both in vitro and in vivo at theconcentration below 100 uM. And when the concentration of withangulatin Awas higher then 100 uM,the interactions between DNA retardation assaymethod. The dose response of withangulatin A was nearly the same to thatof mAMSA and VM-26, and the ED50 value of withangulatin A was 50 nM on rat9L cell. The concentration of withangulatin A needed to inhibit 50% DNAsynthesis detected by thymidine incoporation was about 2 uM.(5) The fundamental characters of anti-cancer drug enhancing topoisomeraseII mediated DNA damage.DNA intercalating, ATP analogue, DNA minor groove binding or topoisomeraseII binding reagents were tested about their inhibitory effect on the mAMSAand VM-26 induced topoisomerase II mediated DNA damage. It was found thatthe amount of damaged DNA induced by mAMSA (which itself is a DNAintercalating agent) was reduced significantly when the DNA intercalatingreagent, such as actinomycin D and acridine orange, was present in thereaction mixture. On the other hand, DNA damage induced by VM-26 wasreduced more dramatically by DNA minor groove binding reagent (such asHOECHST and chromomycin A3) and also by coumermycin A1 and novobiocin--theATPase inhibitors of gyrase. Both the amount of topoisomerase II mediatedDNA damage induced by mAMSA and VM-26 were reduced in the presence ofNPCLHAB3--a DNA binding drug, or withangulatin A--a drug blocking theinteractions between DNA and topoisomerase II by its significanttopoisomerase II bjinding activity. These observations suggest us that ananti-cancer drug enhancing topoisomerase II mediated DNA damage shouldhave both the DNA binding and topoisomerase II binding characters togetherin favor of the formation of the DNA-drug-enzyme ternary complex.我們由豬脾臟中分離出拓樸異構□Ⅱ,含有二種polypeptides,分子量各為160kda及150kda,雖然這二個polypeptides,其in situ peptide mapping之pattern不同。但是由protein kinase c,或myelin basic protein kinase作32p labeling所產生具放射性之peptide的patterns卻完全一樣。由這二個poly-peptides所產生的抗體ab160和ab150都會抑制dna relaxation反應,而且這二種抗體分別認識human拓樸異構□Ⅱ之不同domains。利用所純化出來的末拓樸異構□Ⅱ,我們探討拓樸異構□Ⅱ本身及其相關性質之一系列研究。結果為:(一).拓樸異構□Ⅱ之 dna dependent atpase 活性和 atp 本身濃度有關。當[atp]< 0.2 m 時,atp 水解之 km 值為 0.01 mm, vm 值為 4.5x10-4 mm/min。而當atp 濃度大於 0.2 mm 時, km 值和 vm 值均變大。抗癌藥物 vm-26和withangulatin a 均會 uncouplung 拓樸異構□Ⅱ之 atp 水解與 dna strand passage。(二).利用所純化之拓樸異構□Ⅱ,我們探討一段含有 colicin sl ori 及 humanhsp70基因5' 端之 2.3 kb dna 中拓樸異構□Ⅱ之 cutting sites。利用 sangermethod (dideoxy dna sequencing mrthod) 作 stantard, 我們找到並定出12個cutting sites 之位置,這 12 個 cutting sites 中有 10 個為 A。(三).接著我們應用所純化出來的拓樸異構□Ⅱ,以是否會引起 topo Ⅱmediated dnadamage之特性去飾選作用在其上的抗癌中藥成分。得到withangulatin A。withangulatin a 在低濃度範圍(0.5-1 & 10-20 u m)時促進 topoⅡmediated dnadamage; 但高濃度時,withangulatin a 則會阻礙拓樸異構□Ⅱ和 dna 的結合。這可能和 withangulatin a 對大白鼠 9L細胞的ED50值和MAMSA及VM-26INO ED50值相當,約為50nM。而withangulatin A 對dna合成的抑制則較vm-26緩和, 50%抑制值為2um。(四).利用會和dna 結合, 或會和拓樸異構□Ⅱ之atp binding domain(s)結合的reagents, 測試它們對topo Ⅱ mediated dna damage 反應的影響, 發現具dnaintercalating 性質之reagents (例如; actinomycin d 及 acridine orange) 對同樣是具 dna interacting 性質之 mamsa 所引起的topo Ⅱmediated dna damage 的抑制較強, 而結合到dna minor groove 之reagent (例如; chromomycin a3及hoechst 33342) 對同樣結合到dna minor groove 之 vm-26 所引起之topo Ⅱmediated dna damage 之抑制較強。而可以阻止拓樸異構□Ⅱ與dna 結合之高濃度withangulatin a 則對mamsa 及 vm-26所引起的topo Ⅱ mediated dna damage 的能力相近。反應液中再加入red-120 dye 可以使由這些抗癌藥物所引起的topo Ⅱmediated 之 broken dna重回 reunioned form。