HL-60 세포의 유전자 발현 및 topoisomerase의 기능 활성에 미치는 억제제의 영향

정인철(In Cheol Jeong),조무연(Moo Youn Cho),박장수(Jang Su Park) 한국생명과학회 2008 생명과학회지 Vol.18 No.1

인체 DNA topoisomerase는 DNA를 단일 또는 두 가닥을 일시적인 절단을 촉매하여 DNA의 topological 문제를 조절함으로써, DNA 복제, 전사, 재조합과 유사분열 과정 등에 관여한다. 이 효소는 많은 항생, 항암제의 표적효소로서 널리 알려져 있으며, 이들 유도체를 이용한 다양한 억제제의 개발과 임상적 응용에 관한 연구가 활발하게 진행되고 있다. 본 실험에서는 인체 백혈병 HL-60 세포에서 topoisomerase 억제제가 topoisomerase 기능 활성과 유전자 발현을 조절하는지를 규명하기 위하여 본 연구를 수행하였다. 연구 방법은 HL-60세포에 topoisomerase type Ⅰ과 type Ⅱ의 대표적 억제제인 10-hydroxycamptothecin (10-CPT)과 doxorubicin을 투여한 후 total RNA를 분리하였고, 10K-oligonucleotide microarray 방법으로 분석하여 유전자의 발현 양상을 조사하였다. 연구 결과에 의하면 10-CPT 또는 doxorubicin을 투여한 HL-60세포에서의 유전자 발현 양상은 주로 signal transduction, cell adhesion, cell cycle, cell growth, cell proliferation, cell differentiation, transcription 및 immune response 등과 관련이 있었다. Topoisomerase type Ⅰ의 억제제인 10-CPT를 HL-60 세포주에 투여 하였을 때 type Ⅰ으로 분류되는 topoisomerase Ⅲα, Ⅲβ 및 Ⅰ의 발현은 증가하였으나 type Ⅱ인 topoisomerase Ⅱα와 Ⅱβ의 유전자의 발현은 감소되었다. 반대로 type Ⅱ의 억제제인 doxorubicin을 투여하였을 때는 앞의 결과와 상반된 topoisomerase Ⅱα와 Ⅱβ의 유전자의 발현이 현저히 증가되었으며, topoisomerase Ⅲα와 Ⅲβ의 mRNA의 발현은 약간 감소하는 양상을 보였으나 의미 있는 차이는 없었다. 이 연구 결과는 앞으로 항암제의 기전을 밝히고 약물에 대한 치료 반응을 예측하고 새로운 약제 개발에 기초자료가 될 것으로 여겨진다. This studies were designed to elucidate whether inhibitors of topoisomerase regulate function and activity of topoisomerase, and gene expression in HL-60 human leukemia cells. HL-60 cells were treated with 10-hydroxycamptothecin or doxorubicin, total RNA was isolated, and expressed genes were investigated with human oligonucleotide microarray containing 10K gene, respectively. Expression profiles of the human leukemia HL-60 cells treated with 10-hydroxycamptothecin (10-CPT) or doxorubicin associated with signal transduction, cell adhesion, cell cycle, cell growth, cell proliferation, cell differentiation, transcription and immune response, especially genes related with transcription and cell growth. In HL-60 cells treated with 10-CPT, the expression of topoisomerase Ⅲα, Ⅲβ and Ⅰ gene from oligo chip microarray analysis were increased over, but the expression of topoisomerase IIα and IIβ gene were decreased over. In contrast, the expression of topoisomerase Ⅱα and Ⅱβ gene were increased over in HL-60 cells treated with doxorubicin, whereas the expression of topoisomerase Ⅲα and Ⅲβ mRNA remained no significant change. These results suggest that these data may be useful for novel therapeutic markers.

Doxorubicin이 HL-60 사람 백혈병 세포내 topoisomerase의 활성과 발현에 미치는 영향

조무연,정인철 고신대학교 의학부 2004 高神大學校 醫學部 論文集 Vol.19 No.1

Background DNA topoisomerases are essential enzymes present in all organisms. They modify DNA topology in connection with a number of nuclear processes, such as replication, transcription, chromatin remodeling, chromatin condensation/decondensation, recombination and repair. In the strand-breakage reaction by a DNA topoisomerase, a tyrosyl oxygen of the enzyme attacks a DNA phosphorus, forming a covalent phosphotyrosine link and breaking a DNA phosphodiester bond at the same time. DNA topoisomerase have been shown to be the molecular targets of many antimicrobial and anticancer agents. Among topoisomerase-targeting drugs in clinical use at present, most act by trapping the covalent DNA-enzyme intermediates to convert a normal cellular enzyme to a DNA damaging agent. This studies were designed to elucidate whether pretreatment of HL-60 human leukemia cells with the topoisomerase I - directed drug doxorubicin would increase the expression of topoisomerase and to investigate the activity of topoisomerase mediated by doxorubicin in nuclear extract from HL-60 human leukemia cells. Methods We have conducted experiments on topoisomerase assay using gel electrophoresis, pUC-X I cloning, DNA sequencing, cell cytotoxicity in drug-treated cells, topoisomerase purification, quantitative RT-PCR analysis, northern blotting techniques, respectively. Results Doxorubicin inhibited the relaxation activity of topoisomerase in pUC19 DNA at warious concentrations (0.4-50 μM), while it enhanced the cleavage of topoisomerase in the pUC-X I by forming a cleavable complex at 0.4-2μM. The levels of the topoisomerase IIα mRNA from RT-PCR analysis and northern blot were increased in HL-60 cells treated with doxorubicin, whereas the expression of topoisomerase I and IIβ mRNA from RT-PCR analysis remained no significant change. Conclusion Our results suggest that overexpression of topoisomerase IIα mRNA by topoisomerase II-directed drug treatment is due to stabilizing drug-enzyme-DNA "cleavable complex".

Doxorubicin이 HL-60 사람 백혈병 세포내 topoisomerase의 활성과 발현에 미치는 영향

조무연,정인철 고신대학교(의대) 고신대학교 의과대학 학술지 2004 고신대학교 의과대학 학술지 Vol.19 No.1

Background : DNA topoisomerases are essential enzymes present in all organisms. They modify DNA topology in connection with a number of nuclear processes, such as replication, transcription, chromatin remodeling, chromatin condensation/decondensation, recombination and repair. In the strand-breakage reaction by a DNA topoisomerase, a tyrosyl oxygen of the enzyme attacks a DNA phsphorus, foriming a covalent phosphotyrosine link and breaking a DNA phosphodiester bond at the same time. DNA topoisomerases have been shown to be the molecular targets of many antimicrobial and anticancer agents. Among topoisomerase-targeting drugs in clinical use at present, most act by trapping the covalent DNA-enzyme intermediates to convert a normal cellular enzyme to a DNA damaging agent. This studies were designed to elucidate whether pretreatment of HL-60 human leukemia cells with the topoisomerase 1-directed drug doxorubicin would increase the expression of topoisomerases and to investigate the activity of directed drug doxorubicin would increase the expression of topoisomerases and to investigate the activity of topoisomerase mediated by doxorubicin in nuclear extract from HL-60 human leukemia cells. Methods : We have conducted experiments on topoisomerase assay using gel electrophoresis, pUC-X 1 cloning, DNA sequencing, cell cytotoxicity in drug-treated cells, topoisomerase purification, quantitative RT-PCR analysis, northern blotting techniques, respectively. Results : Doxorubicin inhibited the relaxation activity of topoisomerase in pUC19 DNA at various concentrations (0.4-50uM), while it enhanced the cleavage of topoisomerase in the pUC-X 1 by forming a cleavable complex at 0.4-2uM. The levels of the topoisomerase 2a mRNA from RT-PCR analysis and northern blot were increased in HL-60 cells treated with doxorubicin, whereas the expression of topoisomerase 1 and 2b mRNA from RT-PCR analysis remained no significant change. Conclusion : Our results suggest that overexpression of topoisomerase 2a mRNA by topoisomerase 2-directed drug treatment is due to stabilizing drug-enzyme-DNA "cleavable complex".

Mycoplasma Hominis에서 분리한 Type II-DNA Topoisomerase의 효소반응 특성

정인철,박인달 고신대학교의과대학 2008 고신대학교 의과대학 학술지 Vol.23 No.4

Background : DNA topoisomerases are essential enzymes that catalyze the mutual conversion of the topological states of DNA. Two major classes of enzyme have been isolated from a number of organisms. Nevertheless, the enzymatic properties of DNA topoisomerases in the mycoplasma species has not been identified yet. Methods : We have examined the activities of the DNA topoisomerases from various types of mycoplasma and the characterizations of partially purified type II-topoisomerase fractions from Mycoplasma hominis. Results : The levels of the DNA topoisomerase activities in Mycoplasma hominis and Mycoplasma fermentans were higher than those in Ureaplasma urealyticum, Mycoplasma penetrans and Mycoplasma pneumoniae. DNA topoisomerase has been partially purified from Mycoplasma hominis by Ultrogel AcA34 gel filtration. This enzyme was highly stimulated in relaxation of supercoiled DNA by ATP-Mg2+. Topoisomerase mediated DNA cleavage was enhanced by mAMSA (eukaryotic type II inhibitor) and norfloxacin (prokaryotic type II inhibitor) at various concentrations (0.05-0.5 mM). Conclusion : Our results suggest that enzymatic properties of type II-topoisomerase in Mycoplasma hominis have analogous to type II-human DNA topoisomerase and type IIA-bacterial topoisomerase.

HL-60 사람 백혈병 세포에서 irinotecan이 topoisomerase Ⅰ과 Ⅲ의 활성과 발현에 미치는 영향

정인철,조무연 KOSIN UNIVERSITY COLLEGE OF MEDICINE 2006 高神大學校 醫學部 論文集 Vol.21 No.1

Background DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. DNA topoisomerases have been shown to be the molecular targets of many antimicrobial and anticancer agents. Among topoisomerase- targeting drugs in clinical use at present, most act by trapping the covalent DNA-enzyme intermediates to convert a normal cellular enzyme to a DNA damaging agent. This studies were designed to elucidate whether pretreatment of HL-60 human leukemia cells with the topoisomerase Ⅰ - directed drug irinotecan would increase the expression of topoisomerases and to investigate the activity of topoisomerase mediated by irinotecan in nuclear extract from HL-60 human leukemia cells. Methods We have conducted experiments on cell cytotoxicity in drug-treated HL-60 cells, topoisomerase purification, topoisomerase assay using gel electrophores and oligo chip microarray analysis, respectively. Results Irinotecan inhibited the relaxation activity of topoisomerase in pUC19 DNA at various concentrations (range 0.4-50 μM). Irinotecan IC(50) values was 1 uM for HL-60 cells. The levels of the topoisomerase Ⅰ(type IB) Ⅲ(type IA) mRNA remained no significant change from oligo chip microarray analysis. Conclusion Our results suggest that topoisomerase I protein is one of the cellular targets of irinotecan but this drug does not coordinate regulation with type Ⅰ topoisomerase mRNA levels.

HL-60 사람 백혈병 세포에서 irinotecan이 topoisomerase I과 DI의 활성과 발현에 미치는 영향

정인철,조무연 고신대학교(의대) 고신대학교 의과대학 학술지 2006 고신대학교 의과대학 학술지 Vol.21 No.1

Background DNA topois아nerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. DNA topoisomerases have been shown to be the molecular targets of many antimicrobial and anticancer agents. Among topoisomerase- targeting drugs in clinical use at present, most act by trapping the covalent DNA-enzyme intermediates to convert a normal cellular enzyme to a DNA damaging agent. This studies were designed to elucidate whether pretreatment of HL-60 human leukemia cells with the topoisomerase I - directed drug irinotecan would increase the expression of topoisomerases and to investigate the activity of topoisomerase mediated by irinotecan in nuclear extract from HL-60 human leukemia cells. Methods We have conducted experiments on cell cytotoxicity in drug-treated HL-60 cells,topoisomerase purification, topoisomerase assay using gel electrophores and oligo chip microarray analysis,respectively. Results Irinotecan inhibited the relaxation activity of topoisomerase in pUC19 DNA at various concentrations (range 0.4-50 ᄍ M〉, Irinotecan IC(50) values was 1 uM for HL-60 cells. The levels of the topoisomerase Ktype IB) and IlKtype IA) mRNA remained no significant change from oligo chip microarray analysis. Conclusion Our results suggest that topoisomerase I protein is one of the cellular targets of irinotecan but this drug does not coordinate regulation with type I topoisomerase mRNA levels.

HL-60 사람 백혈병 세포로부터 topoisomerase type 1B의 동정

정인철,곽충근,조무연 고신대학교(의대) 고신대학교 의과대학 학술지 2004 고신대학교 의과대학 학술지 Vol.19 No.1

Background : DNA topoisomerases fall into two categories -type 1 and type 2. For the type 1 enzymes, the DNA strands are transiently broken on at a time; for the type 2 enzymes, by contrast, a pair of strands in a DNA double helix are transiently broken in concert by a dimeric enzyme molecule. The two types can be further divided into four subfamilies: 1A, 1B, 2A and 2B. DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. Camptothecin is an antitumor alkaloid that has been isolated from the Chinese tree, Camptotheca acuminata. We have developed a procedure for the simultaneous purification of type 1 and 2 topoisomerase from HL-60 human leukemia cells, and identified type 1B topoisomerase. Methods : DNA topoisomerase was purified from HL-60 human leukemia cells by a simple and fast four-step procedure : seletive ammonium sulfate precipiation, chromatography on Ultrogel A6 and DNA- cellulose, followed by ultracentrifugation on a glycerol gradient. Enzyme activity, and molecular weight were assayed by measuring the relaxation of supercoiled pUC19 DNA using agarose gell elctrophoresis and sodium dodesyl sulfate denaturing gel electrophoresis, respectively. Results Type 1 and 2 topoisomerases were isolated from extracted nucleoprotein complexes by glycerol gradient centrifugation, respectively. Human type 1B topoisomerase was purified 8.6-fold as compared to the whole cell homogenate, with 12% yield. The purified type 1 topoisomerase has a molecular weight of 100 kDa as determined by SDS polyacrylamide gel electrophoresis. The enzyme relaxes supercoiled DNA in the absence of ATP or Mg2+. Camptothecin inhibited the relaxation activity of type 1 topoisomerase in pUC19 DNA by forming a cleavable complex at various concentrations (0.4-50 uM). Conclusion : The results showed that a type 1B topoisomerase has been purified to near homogeneity from HL-60 human leukemia cells, and partial purification of type 2A from small samples was achieved by isolation of cell nuclei. Thus, the potential antitumor activity of a type 1 and 2 topoisomerase-targeted drug might be easily screened by testing the drug's ability to cause DNA cleavage in the presence of enzymes.

A novel epoxypropoxy flavonoid derivative and topoisomerase II inhibitor, MHY336, induces apoptosis in prostate cancer cells

Patra, Nabanita,De, Umasankar,Kang, Jin-Ah,Kim, Ji Mim,Ahn, Mee Young,Lee, Jaewon,Jung, Jee H.,Chung, Hae Young,Moon, Hyung Ryong,Kim, Hyung Sik Elsevier 2011 european journal of pharmacology Vol.658 No.2

<P><B>Abstract</B></P><P>Here, we reported the synthesis of a novel topoisomerase II inhibitor, MHY336, which that has strong topoisomerase-mediated anticancer activity but fewer side effects than other topoisomerase II inhibitors. The catalytic activity of MHY336 on the topoisomerase II enzyme was the same as that of the etoposide. In a cell-free system, MHY336 exhibited a potent activity on scavenging of reactive oxygen species against 3-morpholinosydnonimine hydrochloride (SIN-1)-induced oxidative stress. An <I>in vitro</I> cell-based assay demonstrated that MHY336 significantly inhibited the proliferation of three prostate cancer cell lines, LNCaP, PC-3, and DU145 cells. Notably, the cytotoxicity of MHY336 was more potent in LNCaP cells (IC<SUB>50</SUB>=1.39μM) than in DU145 (IC<SUB>50</SUB>=2.94μM) and PC3 cells (IC<SUB>50</SUB>=3.72μM). Furthermore, MHY336 treatment induced similar levels of cytotoxicity compared to doxorubicin treatment (IC<SUB>50</SUB>=1.55μM) in LNCap cells. Also, MHY336 significantly down-regulated topoisomerase II alpha expression and up-regulated p53 expression in LNCaP cells (wild-type p53), whereas it up-regulated the topoisomerase II alpha protein in both DU145 and PC3 cells (p53 mutated or deleted). MHY336 induced G2/M or S phase arrest in LNCaP cells through a well-documented topoisomerase II-dependent mechanism. Further studies using Annexin V-FITC binding assay, DAPI staining, and Western blot analyses illustrated that MHY336 markedly induced apoptotic cell death via the mitochondria-mediated intrinsic pathway in LNCaP cells. These results suggest that MHY336 is an attractive chemotherapeutic agent because of its topoisomerase II-mediated anti-tumour activity in human prostate cancer.</P>

HL-60 사람 백혈병 세포로부터 topoisomerase type IB의 동정

정인철,곽충근,조무연 고신대학교 의학부 2004 高神大學校 醫學部 論文集 Vol.19 No.1

Background DNA topoisomerases fall into two categories - type Ⅰand typeⅡ. For the type I enzymes. the DNA strands are transiently broken one at a time; for the type II enzymes. by contrast, a pair of stands in a DNA double helix are transiently broken in concert by a dimeric enzyme molecule. The two types can be further divided into four subfamilies : IA, IB, IIA and IIB. DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. Camptothecin is an antitumor alkaloid that has been isolated from the Chinese tree. Camptotheca acuminata.. We have developed a procedure for the simultaneous purification of type I and II topoisomerase from HL-60 human leukemia cells, and identified type IB topoisomerase. Methods DNA topoisomerase was purified from HL-60 human leukemia cells by a simple and fast four-step procedure : selective ammonium sulfate precipitation, chromatography on Ultrogel A6 and DNA-cellulose. followed by ultracentrifugation on a glycerol gradient. Enzyme activity, and molecular weight were assayed by measuring the relaxation of supercoiled pUC19 DNA using agarose gel electrophoresis and sodium dodesyl sulfate denaturing gel electrophoresis, respectively, Results Type I and II topoisomerases were isolated from extracted nucleoprotein complexes by glycerol gradient centrifugation, respectively. Human type IB topoisomerasewas purified 8.6-fold as compared to the whole cell homogenate, with 12% yield. The purified type I topoisomerase has a molecular weight of 100 kDa ad determined by SDS polyacrylamide gel electrophoresis. The enzyme relaxes supercoiled DNA in the absence of ATP of Mg2+. Camptothecin inhibited the relaxation activity of type I topoisomerase in pUC19DNA by forming a cleavable complex at various concentratrions (0.4-50 μM). Conclusion The results showed that a type IB topoisomerase has been purified to near homogeneity from HL-60 human leukemia cells, and partial purification of type IIA from small samples was achieved by isolation of cell nuclei. Thus, the potential antitumor activity of a type I and II topoisomerase-targeted drug might be easily screened by testing the drug's ability to cause DNA cleavage in the presence of enzymes.

1-(2-furyl)-3-phenylpropenone 유도체의 DNA Topoisomerase I 저해활성에 대한 parameter focusing

명평근(Pyung Keun Myung),최수라(Su La Choi),성낙도(Nack Do Sung) 대한약학회 2000 약학회지 Vol.44 No.4

Parameter focusing on the DNA topoisomerase-I inhibition with X-substituted phenyl substituents in 1-(2-furyl)-3-phenylpropenone derivatives as inhibition material were analyzed. From the basis on the results, the inhibition on DNA topoisomerase I suggested that the inhibition activities of X-substituted phenyl substitutents would depend largely on the net charge of beta-carbon atom, LUMO energy (e.v.) and STERIMOL parameter, B5 (width) of X. Among them, non-substituent (X=H), 1 and 2,2-dichloro substituent, 4 showed the highest DNA topoisomerase-I inhibition activity.