Cancer Research and Treatment

Original Articles

Role of ATF on Transcriptional Regulation of DNA Topoisomerase II a Gene in HL - 60 Arrested to G2 / M and M Phase: Kyu Lim, Mee Young Son, Byung Ik Choi, Kyung Ah Yun, Meizi Zheng, Tae Wook Kang, Young Chul Lee, Jong II Park, Wan Hee Yoon, Byung Doo Hwang; J Korean Cancer Assoc. 1999;31(6):1279-1287.

Abstract PDF: PURPOSE
To gain insight on transcriptional repression of Topo II a in HL-60 cells arrested to G2/M and M phase, the levels of Topo IIa mRNA and the binding activity of ATF have been investigated with Northern blot hybridization and DNA mobility shift assay, respectively.
MATERIALS AND METHODS
HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-mactivated fetal bovine serum and antibiotics in a humidified 5% CO2 at 37C degree. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. A Xho I-Mlu I fragment of phTOP2 was used as probe for Northern blot analysis of Topo II a mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA oligomer (upper strand, 5-TCTCCGCTATGACGCCGAGTGGTG-3) for ATF binding activity was mixed with nuclear extracts in a 20 pl reaction volume containing 60 mM KC1, 12 mM HEPES, pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 0.2 mM DTT, 12% glycerol, and 2 ug of poly [dI-dC].
RESULTS
HL-60 cells were arrested at G2/M phase and M phase after taxol or nocodazole treatment. The levels of Topo II a mRNA were reduced at 24 hours after exposure with nocodazole or taxol but the unknotting activities were not changed. DNA mobility shift assay using oligonucleotide containing the ATF binding site showed that ATF binding activity was reduced after pretreatment of nododazole or taxol.
CONCLUSIONS
These results suggest that the reduction of ATF binding activity may be important to transcriptional repression of Topo II a gene by nocodazole and taxol in HL- 60 cells.

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Studies on the H2B Histone , c-Myc and DNA Topoisomerase 1 Gene Expression during HL - 60 Differentiation by All - Trans Retinoic Acid: Kyu Lim, Jeong Dong Park, Byung Han Choi, Young Jin Lee, Kye Young Kim, Myung Sun Lee, Eun Mi Chang, Sam Yong Kim, Gi Ryang Kweon, Sang Tae Kwak, Byung Doo Hwang; J Korean Cancer Assoc. 1994;26(2):285-296.

Abstract PDF: Effects of all-trans retinoic acid(retinoic acid) on DNA replication, H2B histone and DNA topoisopmerase I(Topo I) gene expression have been investigated in human promyelocytic leukemia cell line HL-60. DNA synthesis decreased at 24 hours after exposure of retinoic acid in the HL-60 cells. H2B histone mRNA rapidly reduced at 48 hours and Topo I and c-myc mRNA at 24 hours in retinoic acid-exposured HL-60 cells, respectively. The levels of c-myc, H2B his- tone and Topo I gene expression were reduced in proportion to the concentration of retinoic acid. The H2B histone mRNA in retinoic acid-exposured cells was elevated by cycloheximide treatment, while the level of Topo I mRNA was constant. These results suggest that regulations of H2B histone, c-myc and Topo I gene expression are different from one another, and repression of Topo I gene is closely correlated with c-myc gene during retinoic acid-induced differentiation of HL-60 cells.

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Alterations in DNA Cleavage Site of Topoisomerase 2 by Benzo(a)pyrene , m-AMSA , Ellipticine and Doxorubicin in Human c-myc Protooncogene: Sang Wook Lee, In Cheol Jeong, Moo Youn Cho; J Korean Cancer Assoc. 1996;28(3):520-533.

Abstract PDF: The nuclear enzyme DNA topoisomerase II, involving in DNA replication and transcription, mediates DNA scission in cells exposed to certain classes of intercalating agents, and is trapped as a covalent protein-DNA complex. In a previous study, we have found that benzo(a)pyrene(BP) and it's metabolites specifically bind to the DNA topoisomerase II fraction of mouse fibroblast C3H/10Tl/2 cell cultures. The enzyme was predicted to be a primary target of BP, causing malignancy. We therefore investigated in the present study changes in DNA cleavage by various concentrations of BP bound to DNA topoisomerase II in the human c-myc protooncogene. At the same time, the effects of m-AMSA(amsacrine), ellipticine and doxorubicin were also investigated. DNA topoisomerase II was purified by glycerol gradient centrifugation from mouse leukemia L1210 cells which easily form pro tein-DNA cross linkage. In the experiments of agarose gel electrophoresis which analyzed genomic changes in the human c-myc DNA, the pBR322 DNA was completely reversed from a supercoiled to a relaxed form in the presence of 160ng of purified DNA topoisamerase II. In BP-treated Hind III/Xba I cutting c-myc DNA, DNA cleavage was most apparently increased at 1.7 kb site, the effect being particularly pronounced with 0.1 ￥i M BP. Genomic changes by drug-induced DNA topoisomerase II in the labelled with P32 to restricted c-myc DNA were enhanced predominantly in the cleavage sites of P2 promoter region with m-AMSA, ellipticine and doxorubicin, and in the upstream of exon 1 with BP. In in vivo experiments, in which the effect of BP exposure of in human lymphoblast NC- 37 cells was tested, there was no change in c-myc RNA expression by 0.25 ￥ig/ml of BP. However, the c-myc RNA expression increased significantly with BP of 2.5 ￥ig/mL A simi lar increase in RNA expression was observed in Burkitt lymphoma cells. These resu1ts indicate that an increase of DNA cleavage or increased cleavage at specific sites induce gene expression of abnormal pattern.

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Expression of Multidrug Resistant Genes in Bone Marrow Mononuclear Cells of Patients with Myeloid Leukemia: Seok Goo Cho, Il Ho Yang, Hyeon Seok Eom, Chang Gi Min, Hee Je Kim, Dong Wook Kim, Jong Wook Lee, Chi Wha Han, Woo Sung Min, Won Il Kim, Chun Choo Kim; J Korean Cancer Assoc. 1999;31(1):153-164.

Abstract PDF: PURPOSE
Multidrug resistance mediated by several drug resistant genes impedes the successful outcome of anti-cancer chemotherapy. In this study, we investigated the expressions of drug resistant genes encoding multidrug resistance (MDR1), multidrug resistance-associated protein (MRP), topoisomerase I (Topo I), topoisomerase II g (Topo II a) in narmal volunteers (n=12) in and patients with myeloid leukemia (n=34). Material and Method: We compared the levels of their transcripts in bone matrow mononuclear cells by semiquantitative RT-PCR. The amount of specific transcripts was represented as the optical density ratio of PCR product of target gene to that of B2- microglobulin (MG). Twenty patients of acute myelogenous leukemia (eight in remission state, twelve in refractory) and fourteen patients of chronic myelogenous leukemia (nine in chronic phase and five in blastic crisis) were examined. Twelve normal healthy persons were compared with leukemic patients.
RESULTS
The expression levels of all resistant genes in normal volunteers were relatively high as those of AML patients. Regardless of the disease status including remission status of AML (complete remission versus refractory) and the phase of CML (chronic phase versus blastic phase), the expression levels of all resistant genes in patients with CML were significantly lower than in the patients with AML (p < 0.05). Of interest, the patients with refractary AML did not show any statistical difference in comparison with normal controls and even the patients with AML in complete remission. Among the four drug resistant genes, the optical density ratio of MDRl was significantly lower than that of any other genes (p<0.05). Using HL-60 cell line, we compared the changes of various resistant gene expressions before and after differentiation induced by dimethylsulfoxide. The expressions of resistant genes declined in paralle1 with granulocytic differentiation, suggesting that the induction of cell differentiation might make leukemic cells susceptible to chemotherapeutic agents.
CONCLUSION
It is impossibble to explain the mechanism of drug resistance by comparing the level of drug resistant gene expression between nonnal subjects and patients with myeloid leukemias. Therefore, we suppose that longitudinal study of drug resistant gene expression is necessary to demonstrate the development of drug resistant during chemotherapy.

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Expression of DNA Topoisomerase II and P-Glycoprotein in Breast Cancer: Hye Rim Park, Eun Ha Jung, Jin Hee Sohn, Young Euy Park; J Korean Cancer Assoc. 1997;29(2):243-249.

Abstract PDF: PURPOSE
To determine whether the expression of DNA topoisomerase II and P-glycoprotein are of prognostic value. MATERIALS AND METHODS: We evaluated the expression of DNA topoisomerase II and P-glycoprotein immunohistochemically in a retrospective study of samples from 44 patients with breast cancer. Thirty two among 44 patients (72.7%) received chemotherapeutic treatments (CMF or FAC protocol) and/or tamoxifen postoperatively.
RESULTS
P-glycoprotein was detected in the 27 samples of 44 patients (61.3%). The expression of P-glycoprotein was increased in the patients older than 50 years, with distant metastases, and with death on follow-up. DNA topoisomerase II was detected in the 34 samples of 44 patients (77.2%). The expression of topoisomerase II was increased in the patients younger than 50 years, with recurrent tumor, with distant metastases, and with death on follow-up. The expression of P-glycoprotein and topoisomerase II was not correlated with other clinico-pathological factors including the size of primary tumor, involvement of lymph node, histologic grade, and clinical stage. The correlation between expression of P-glycoprotein and topoisomerase II was not significant.
CONCLUSION
The immunohistochemical evaluation of P-glycoprotein and topoisomerase II before treatment in breast cancer has little clinical prognostic value.

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