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DNA topoisomerase IIalpha expression and the response toprimary chemotherapy in breast cancer.

ABSTRACT: The alpha isoform of Topoisomerase IIalpha (Topo IIalpha) is a proliferation marker as well as a target for several chemotherapeutic agents such as anthracyclines. In vitro studies have demonstrated the relationship between the Topo IIalpha expression level and chemosensitivity of target cancer cells. To verify this effect in vivo, we selected 125 patients presenting with T(2)>3 cm and T(3) N(0-1) M(0) breast tumours who were treated by six cycles of primary chemotherapy, including epirubicin before any surgery. Therapy response was assessed by clinical and X-ray mammogram measurements of tumour shrinkage. The pretherapeutic core biopsies were immunostained with a monoclonal antibody (Ki-S7) against Topo IIalpha. Ki-S7 positivity ranged from 0 to 50% (median, 15%). A high percentage of Ki-S7-positive cells (>15%) was associated with tumour regression under chemotherapy (OR=2.88, CI: 1.3-6.4, P=0.004). Ki-S7 further emerged as an independent predictor of tumour regression (OR=3.34, CI: 1.41-7.93, P=0.006), together with tumour size of less than 40 mm (OR=3.82, CI: 1.58-9.25, P=0.002) and negative oestrogen receptor (ER) status (OR=3.35, CI: 1.43-7.86, P=0.005), in a multivariate analysis including tumour size, SBR grade, ER and PR status, Ki-67, p53 and Her-2/neu. Our clinical results confirm in vitro data on the relationship between Topo IIalpha expression and tumour chemosensitivity and thus may have important practical implications.

SUBMITTER: MacGrogan G

PROVIDER: S-EPMC2376904 | biostudies-other | 2003 Aug

REPOSITORIES: biostudies-other

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DNA topoisomerase IIalpha and -beta expression in human ovarian cancer.

Project description:To study DNA topoisomerase IIalpha (Topo-IIalpha) and -beta expression and regulation in human ovarian cancer, 15 ovarian tumour samples were investigated. To compare different levels of expression, the samples were screened for topo IIalpha and -beta mRNA with Northern blotting and a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay for Topo-IIalpha mRNA. Additionally, protein levels were determined with Western blotting and topoisomerase II activity levels with the decatenation assay. The results obtained were compared with each other and with the tumour volume index of the samples. In tumours with a tumour volume index > or = 50%, the mRNA levels (as determined by Northern blotting) and protein levels for each isozyme were in accordance. Additionally, correlations were found between Topo-IIalpha RT-PCR data and Topo-IIalpha Northern blot results, and between Topo-IIalpha RT-PCR data and Topo-IIalpha protein levels. Interestingly, Topo-IIbeta protein levels correlated better with Topo-II activity than Topo-IIalpha protein levels. In eight ovarian cystadenoma samples, no Topo-IIalpha protein could be found. In only three out of eight of these cystadenomas, Topo-IIbeta protein could be detected. These findings suggest that Topo-IIalpha and Topo-IIbeta protein levels are up-regulated in ovarian cancer and may indicate that Topo-IIbeta is an interesting target for chemotherapy in ovarian tumours.

| S-EPMC2362692 | biostudies-other

The iron chelator Dp44mT causes DNA damage and selective inhibition of topoisomerase IIalpha in breast cancer cells.

Project description:Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G(1) cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (gamma-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIalpha (top2alpha) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2alpha knockout cells (top2alpha(+/-)) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2alpha(+/+) or top2beta(-/-) cells. Specificity for top2alpha was confirmed using top2alpha and top2beta small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2alpha. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2alpha activity.

| S-EPMC7322628 | biostudies-literature

Topoisomerase IIalpha-dependent induction of a persistent DNA damage response in response to transient etoposide exposure.

Project description:Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25muM etoposide was highly toxic and initiated a double-stranded DNA damage response as reflected by the recruitment of ATM, MDC1 and DNA-PKcs to gammaH2AX foci. While most DNA breaks were rapidly repaired upon withdrawal of the etoposide treatment, the repair machinery remained engaged in foci for at least 24h following withdrawal. TopoII siRNA ablation showed the etoposide toxicity and gammaH2AX response to correlate with the inability of the cell to correct topoIIalpha-initiated DNA damage. gammaH2AX induction was resistant to the inhibition of DNA replication and transcription, but was increased by pre-treatment with the histone deacetylase inhibitor trichostatin A. These results link the lethality of etoposide to the generation of persistent topoIIalpha-dependent DNA defects within topologically open chromatin domains.

| S-EPMC5527959 | biostudies-literature

Altered expression pattern of topoisomerase IIalpha in ovarian tumor epithelial and stromal cells after platinum-based chemotherapy.

Project description:ObjectiveThe aim of this study was to evaluate the expression of topoisomerase IIalpha (TOP2A) in epithelial and stromal cells of ovarian cancer.MethodsTOP2A expression was analyzed prospectively in normal and tumor epithelial and adjacent stromal cells using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) after laser microdissection (n = 38), RNA in situ hybridization (n = 13), and immunohistochemistry (n = 69).ResultsTOP2A mRNA was detected by RNA in situ hybridization in all ovarian cancer samples, with stronger hybridization signals in tumor epithelial cells compared to adjacent stromal cells. The same expression pattern was found by immunohistochemistry (P = .0001). Very interestingly, specific change was found in recurrent ovarian cancer after platinum-based chemotherapy: TOP2A expression decreased in tumor epithelial cells of recurrent ovarian cancer compared to primary ovarian cancer (P = .056), whereas it increased in tumor-adjacent stromal cells in carboplatin-treated recurrent tumors compared to primary ovarian cancer (P = .023).ConclusionTOP2A mRNA and protein expression in ovarian cancer exhibits specific patterns in tumor epithelial and adjacent stromal cells, which are differentially modulated after platinum-based chemotherapy. These data support the recently discovered importance of the stromal compartment in tumor progression and suggest that tumor stromal cells might be relevant to the development of chemotherapy resistance in ovarian cancer.

| S-EPMC1584288 | biostudies-literature

Topoisomerase IIalpha controls the decatenation checkpoint.

Project description:Topoisomerase II (Topo II) is required to separate intertwined sister chromatids before chromosome segregation can occur in mitosis. However, it remains to be resolved whether Topo II has any role in checkpoint control. Here we report that when phosphorylated, Ser 1524 of Topo IIalpha acts as a binding site for the BRCT domain of MDC1 (mediator of DNA damage checkpoint protein-1), thereby recruiting MDC1 to chromatin. Although Topo IIalpha-MDC1 interaction is not required for checkpoint activation induced by DNA damage, it is required for activation of the decatenation checkpoint. Mutation of Ser 1524 results in a defective decatenation checkpoint. These results reveal an important role of Topo II in checkpoint activation and in the maintenance of genomic stability.

| S-EPMC2712943 | biostudies-literature

Relationship between Topoisomerase 2A RNA Expression and Recurrence after Adjuvant Chemotherapy for Breast Cancer.

Project description:PURPOSE: To perform an exploratory analysis of the relationship between gene expression and recurrence in operable hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-normal breast cancer patients treated with adjuvant doxorubicin-containing chemotherapy. EXPERIMENTAL DESIGN: RNA was extracted from archived tumor samples derived from 378 patients with stage I to III HR-positive, HER2-normal breast cancer and analyzed by reverse transcription-PCR for a panel of 374 genes, including the 21-gene recurrence score (RS). Patients were randomized to receive adjuvant doxorubicin plus cyclophosphamide or docetaxel in trial E2197, with no difference in recurrence seen in the treatment arms. All available recurrent cases were selected plus a nonrecurrent cohort. Cox proportional hazard models were used to identify relationships between gene expression and recurrence. RESULTS: TOP2A expression exhibited the strongest association with increased recurrence risk (P = 0.01), and was significantly associated with recurrence (P = 0.008) in a multivariate analysis adjusted for clinicopathologic features. Elevated TOP2A expression above the median was associated with a 2.6-fold increase (95% confidence interval, 1.3-5.2; P = 0.008) in risk of recurrence if the RS was <18, and a 2.0-fold increase (95% confidence interval, 1.2-3.2, P = 0.003) if there was an intermediate RS of 18 to 30. CONCLUSIONS: In patients with HR-positive, HER2-normal breast cancer, a population known to have a low incidence of TOP2A gene alterations thought to be predictive of anthracycline benefit, there is a range of TOP2A RNA expression that is strongly associated with recurrence after adjuvant anthracyclines, which provides information complementary to RS, indicating that it merits further evaluation as a prognostic and predictive marker. (Clin Cancer Res 2009;15(24):7693-700).

| S-EPMC3396025 | biostudies-literature

Metal ion interactions in the DNA cleavage/ligation active site of human topoisomerase IIalpha.

Project description:Human topoisomerase IIalpha utilizes a two-metal-ion mechanism for DNA cleavage. One of the metal ions (M(1)(2+)) is believed to make a critical interaction with the 3'-bridging atom of the scissile phosphate, while the other (M(2)(2+)) is believed to interact with a nonbridging oxygen of the scissile phosphate. Based on structural and mutagenesis studies of prokaryotic nucleic acid enzymes, it has been proposed that the active site divalent metal ions interact with type II topoisomerases through a series of conserved acidic amino acid residues. The homologous residues in human topoisomerase IIalpha are E461, D541, D543, and D545. To address the validity of these assignments and to delineate interactions between individual amino acids and M(1)(2+) and M(2)(2+), we individually mutated each of these acidic amino acid residues in topoisomerase IIalpha to either cysteine or alanine. Mutant enzymes displayed a marked loss of catalytic and DNA cleavage activity as well as a reduced affinity for divalent metal ions. Additional experiments determined the ability of wild-type and mutant topoisomerase IIalpha enzymes to cleave an oligonucleotide substrate that contained a sulfur atom in place of the 3'-bridging oxygen of the scissile phosphate in the presence of Mg2+, Mn2+, or Ca2+. On the basis of the results of these studies, we conclude that the four acidic amino acid residues interact with metal ions in the DNA cleavage/ligation active site of topoisomerase IIalpha. Furthermore, we propose that M(1)(2+) interacts with E461, D543, and D545 and M(2)(2+) interacts with E461 and D541.

| S-EPMC2782489 | biostudies-literature

Protein expression of DNA damage repair proteins dictates response to topoisomerase and PARP inhibitors in triple-negative breast cancer.

Project description:Patients with metastatic triple-negative breast cancer (TNBC) have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose) polymerase (PARP), a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.

| S-EPMC4361640 | biostudies-literature

Construction, characterization, and complementation of a conditional-lethal DNA topoisomerase IIalpha mutant human cell line.

Project description:DNA Topoisomerase IIalpha (topoIIalpha) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoIIalpha expression can be silenced in all cells by the addition of tetracycline. TopoIIalpha-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoIIalpha fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoIIalpha as an indicator for endogenous topoIIalpha dynamics, our analyses provide new evidence that topoIIalpha plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoIIbeta and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

| S-EPMC532048 | biostudies-literature

Using 3'-bridging phosphorothiolates to isolate the forward DNA cleavage reaction of human topoisomerase IIalpha.

Project description:The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. However, the low level of cleavage at equilibrium and the tight coupling of the cleavage and ligation reactions make it difficult to characterize the mechanism by which these enzymes cut DNA. Therefore, to establish a system that isolates topoisomerase II-mediated DNA scission from ligation, oligonucleotide substrates were developed that contained a 3'-bridging phosphorothiolate at the scissile bond. Scission of these substrates generates a 3'-terminal -SH moiety that is a poor nucleophile relative to the normal 3'-terminal -OH group. Consequently, topoisomerase II cannot efficiently ligate phosphorothiolate substrates once they are cleaved. The characteristics of topoisomerase IIalpha-mediated cleavage of phosphorothiolate oligonucleotides were identical to those seen with wild-type substrates, except that no ligation was observed. This unidirectional accumulation of cleavage complexes provided critical information regarding coordination of the protomer subunits of topoisomerase IIalpha and the mechanism of action of topoisomerase II poisons. Results indicate that the two enzyme subunits are partially coordinated and that cleavage at one scissile bond increases the degree of cleavage at the other. Furthermore, anticancer drugs such as etoposide and amsacrine that strongly inhibit topoisomerase II-mediated DNA ligation have little effect on the forward scission reaction. In contrast, abasic sites that increase levels of cleavage complexes without affecting ligation stimulate the forward rate of scission. Phosphorothiolate substrates provide significant advantages over traditional "suicide substrates" and should be valuable for future studies on DNA scission and the topoisomerase II-DNA cleavage complex.

| S-EPMC2733888 | biostudies-literature

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