Transfection of human topoisomerase IIα into etoposide-resistant cells: transient increase in sensitivity followed by down-regulation of the endogenous gene

Asano, Takeshi; An, Taeha; Mayes, Janice; Zwelling, Leonard A.; Kleinerman, Eugenie S.

doi:10.1042/bj3190307

Cited by 31 publications

(19 citation statements)

References 14 publications

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“…RNA was extracted, electrophoresed, transferred and hybridised with a human Topo IIa gene probe (generous gift of Dr L Liu, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, NJ, USA), Topo IIa cDNA fragment, which was amplified from a single-stranded cDNA library using primer set 5 0 -GGAGAGCAGCAACAAAAACA-3 0 (3953 -3972) and 5 0 -CTTGCTTGTGACTGCTTTCG-3 0 (4484 -4503), b-actin probe (generous gift of Dr Eugenie S Kleinerman, University of Texas, MD Anderson Cancer Center), or human glyceraldehyde 3-phosphate dehydrogenase cDNA (Asano et al, 1996a). RT -PCR was performed according to the manufacturer's instructions (Takara, Ootsu).…”

Section: Northern Blot Analysis and Rt -Pcrmentioning

confidence: 99%

“…Proteins from whole-cell lysate were prepared for SDS -polyacrylamide gel electrophoresis by addition of an equal volume of SDS sample buffer (4% SDS, 0.2 M DTT, 20% glycerol) and boiling for 5 min. Samples were electrophoresed (1 Â 10 5 cells lane À1 ) on 7% SDS -polyacrylamide gels and transferred onto nitrocellulose membranes as described previously (Zwelling et al, 1989;Harris and Hochhauser, 1992;Asano et al, 1996a;Zhang et al, 1999). Blots were probed with human Topo I antibody (TopoGEN, Inc., Columbus, OH, USA) and monoclonal antibodies 8D2, which recognise the M r 170 000 form of the Topo IIa enzyme (kindly provided by Dr A Kikuchi, Laboratory of Medical Micology, Research Institute of Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya).…”

Section: Immunoblot Analysis Of Topo IImentioning

confidence: 99%

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Altered expression of topoisomerase IIα contributes to cross-resistant to etoposide K562/MX2 cell line by aberrant methylation

et al. 2005

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KRN 8602 (MX2) is a novel morpholino anthracycline derivative having the chemical structure 3 0 -deamino-3 0 -morpholino-13-deoxo-10-hydroxycarminomycin hydrochloride. To investigate the mechanisms of resistance to MX2, we established an MX2-resistant phenotype (K562/MX2) of the human myelogeneous leukaemia cell line (K562/P), by continuously exposing a suspension culture to increasing concentrations of MX2. K562/MX2 cells were more resistant to MX2 than the parent cells, and also showed crossresistance to etoposide and doxorubicin. Topoisomerase (Topo) IIa protein levels in K562/MX2 cells were lower of those in K562/P cells on immunoblot analysis and decreased expression of Topo IIa mRNA was seen in K562/MX2 cells. Topoisomerase II catalytic activity was also reduced in the nuclear extracts from K562/MX2 cells when compared with K562/P cells. Aberrant methylated CpG of Topo IIa gene was observed in K562/MX2 cells when compared with the parent line on methylation-specific restriction enzyme analysis. To overcome the drug resistance to MX2 and etoposide, we investigated treatment with 5-Aza-2 0 -deoxycytidine (5AZ), which is a demethylating agent, in K562/MX2 cells. 5-Aza-2 0 -deoxycytidine treatment increased Topo IIa mRNA expression in K562/ MX2 cells, but not in K562/P cells, and increased the cytotoxicity of MX2 and etoposide. Methylated CpG was decreased in K562/ MX2 cells after 5AZ treatment. We concluded that the mechanism of drug resistance to MX2 and etoposide in K562/MX2 cells might be the combination of decreased expression of Topo IIa gene and increased methylation, and that 5AZ could prove to be a novel treatment for etoposide-resistant cell lines, such as K562/MX2.

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Section: Northern Blot Analysis and Rt -Pcrmentioning

confidence: 99%

Section: Immunoblot Analysis Of Topo IImentioning

confidence: 99%

Altered expression of topoisomerase IIα contributes to cross-resistant to etoposide K562/MX2 cell line by aberrant methylation

et al. 2005

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“…5 The catalytic activity of Topo II in mammalian cells is mediated by two isoforms [ie, Topo II ␣ (TOP2A) and Topo II ␤]. 6 In vitro studies [7][8][9][10] have suggested that sensitivity to Topo II inhibitors is dependent on the expression level of TOP2A in target cancer cells. Cells with a low concentration of TOP2A protein are less sensitive to Topo II-inhibiting drugs versus cells containing a high concentration of TOP2A.…”

mentioning

confidence: 99%

Assessment of Topoisomerase II α Status in Breast Cancer by Quantitative PCR, Gene Expression Microarrays, Immunohistochemistry, and Fluorescence in Situ Hybridization

Romero

Martín

Cheang³

et al. 2011

The American Journal of Pathology

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Anthracyclines are frequently used for the treatment of breast cancer and topoisomerase II alpha (TOP2A) is considered to be the molecular target. Numerous studies have evaluated the predictive value of TOP2A using different methodological approaches and inconsistent results have been reported. Indeed, the correlation between techniques for the assessment of TOP2A status has not been well evaluated. In this study, we determined TOP2A status in 61 breast tumor samples by realtime PCR, DNA microarrays, immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH), and then evaluated these results with clinical-pathological features and breast cancer intrinsic subtypes. First, we observed a statistical significant correlation of TOP2A gene expression between real-time PCR and microarrays (Pearson coefficient, 0.816; P < 0.001), and both predicted TOP2A IHC results fairly well (area under the curve > 0.74). In contrast, poor agreement between FISH and IHC data was observed (k: 0.134). Secondly, TOP2A expression was found significantly associated with cell proliferation, and with the highly proliferative Luminal B, Her2-enriched and Basal-like intrinsic subtypes. In conclusion, TOP2A expression in breast cancer was associated with high proliferation and aggressive tumor subtypes and appears to be independent of its amplification status. All of these features should be taken into consideration when assessing the predictive value of TOP2A for anthracycline-based chemotherapy.

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“…In vitro studies have shown that the sensitivity of cancer cells to topo inhibitors is at least partly dependent on the expression of the topo II gene. 48 Drug-sensitive cells express high amounts of topo II whereas drug-resistant cells have low amounts of topo II. [49][50][51] For our study on topo IIa, we chose a collection of MCL cases from 1975 to 1985 for two reasons.…”

Section: Tablementioning

confidence: 99%

Topoisomerase IIα expression in mantle cell lymphoma: a marker of cell proliferation and a prognostic factor for clinical outcome

et al. 2004

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Mantle cell lymphoma (MCL) is a malignant lymphoma associated with a relatively aggressive clinical course and a median overall survival time of 3-4 years. Treatment usually consists of combination chemotherapy, often including topoisomerase (topo) inhibitors such as doxorubicin, etoposide and mitoxantrone. Topo IIa is an enzyme that is needed whenever uncoiling of DNA is necessary during the cell cycle. The enzyme is a marker of cell proliferation. We analyzed the expression of topo IIa in relation to Ki-67 and the clinical outcome in patients with MCL. Biopsy specimens from 95 untreated patients enrolled in two multicenter trials (1975)(1976)(1977)(1978)(1979)(1980)(1981)(1982)(1983)(1984)(1985) were investigated immunohistochemically with monoclonal antibodies against topo IIa (Ki-S4) and Ki-67 (Ki-S5). Patients with low (0-10%) topo IIa expression had a median overall survival time of 49.0 months, compared to 17.0 months for patients with high (more than 10%) topo IIa expression. The Kaplan-Meier analysis showed a significant difference in the overall survival time related to the percentage of topo IIa (Po0.001) and Ki-67 (Po0.001) positive tumor cells. Multivariate Cox regression analysis revealed the expression of topo IIa as the most important prognostic factor (Po0.001) in MCL superior to the international prognostic index (IPI), the Ki-67 index and other clinical characteristics.

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Transfection of human topoisomerase IIα into etoposide-resistant cells: transient increase in sensitivity followed by down-regulation of the endogenous gene

Cited by 31 publications

References 14 publications

Altered expression of topoisomerase IIα contributes to cross-resistant to etoposide K562/MX2 cell line by aberrant methylation

Altered expression of topoisomerase IIα contributes to cross-resistant to etoposide K562/MX2 cell line by aberrant methylation

Assessment of Topoisomerase II α Status in Breast Cancer by Quantitative PCR, Gene Expression Microarrays, Immunohistochemistry, and Fluorescence in Situ Hybridization

Topoisomerase IIα expression in mantle cell lymphoma: a marker of cell proliferation and a prognostic factor for clinical outcome

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