Topo IIalpha Research Articles

Tumor growth-arrest effect of tetrahydroquinazoline-derivative human topoisomerase II-alpha inhibitor in HPV-negative head and neck squamous cell carcinoma

Oral malignancies continue to have severe morbidity with less than 50% long-term survival despite the advancement in the available therapies. There is a persisting demand for new approaches to establish more efficient strategies for their treatment. In this regard, the human topoisomerase II (topoII) enzyme is a validated chemotherapeutics target, as topoII regulates vital cellular processes such as DNA replication, transcription, recombination, and chromosome segregation in cells. TopoII inhibitors are currently used to treat some neoplasms such as breast and small cells lung carcinomas. Additionally, topoII inhibitors are under investigation for the treatment of other cancer types, including oral cancer. Here, we report the therapeutic effect of a tetrahydroquinazoline derivative (named ARN21934) that preferentially inhibits the alpha isoform of human topoII. The treatment efficacy of ARN21934 has been evaluated in 2D cell cultures, 3D in vitro systems, and in chick chorioallantoic membrane cancer models. Overall, this work paves the way for further preclinical developments of ARN21934 and possibly other topoII alpha inhibitors of this promising chemical class as a new chemotherapeutic approach for the treatment of oral neoplasms.

Role of b‐lapachone treatment mediated base excision repair protein PARylation in colorectal cancer cells.

Poly (ADP‐ribose) PAR is a protein post‐translational modification catalyzed by a family of enzymes known as PARPs. Most solid tumors overexpress NAD(P)H;Quinone Oxidoreductase 1 (NQO1), which catalyzes a futile redox cycle of b‐lapachone during exposure, resulting in the formation of high levels of reactive oxygen species (ROS). Futile cycle b‐lapachone recycling produces elevated superoxide levels, resulting in massive levels of hydrogen peroxide that lead to substantial DNA damage and endoplasmic reticulum Ca2+ release. DNA damage and Ca+2 entry into the nucleus leads to the hyperactivation of poly(ADP‐ribose) polymerase 1 (PARP1). A majority of parylation is noted when hyperactivated PARP1 self‐parylates, forming extensive Poly(ADP) ribosylated PAR‐PARP1 levels are noted within 5 mins of exposure to 4 μM β‐lap in A549 cells, as noted by Western blotting using an antibody specific for PARylated moieties. PAR‐PARP1 is a protein post‐translational modification catalyzed by a family of enzymes known as PARPs. DNA single strand breaks along with base‐damage and abasic sites together with nuclear Ca2+ levels lead to hyperactivation of PARP1, a DNA repair enzyme that utilizes NAD+ to generate PAR moieties. This is followed by dramatic NAD+/ATP losses and tumor cells die of a special programmed necrosis process, referred to as NAD+‐keresis. PAR‐PARP1 is rapidly degraded by poly(ADP‐ribose) glycohydrolase (PARG) a exo‐ and endoglycosidase.PARG‐knockdown HCT116 cells were used to gain insight into the function of PARG and whether PARG depletion would enhance b‐lapachone induced cell death by blocking PAR recycling. Proteomic screening after b‐lapachone treatment in colorectal cancer cell line revealed the PARylation landscape of base excision, mismatch and nucleotide excision repair proteins. Twenty (20) of the proteins were found to overlap with H2O2 treatments (200 μM or 2 mM), while one protein (i.e., SPSF) was unique to b‐lapachone treatment. b‐Lapachone treatment affects BER pathways and qualitative analysis of the human Asp‐ and Glu‐ADP‐ribosylated proteome after b‐lapachone treatment indicate major alterations in BER pathways. We confirmed that b‐lapachone mediated cytotoxicity was independent of PARG knockdown or TOPO I or II protein losses. In contrast, RFC1 and PCNA siRNA‐mediated protein knock downs significantly enhanced b‐lapachone lethality, whereas knockdown of Topo I or Topo IIalpha did not affect b‐lapachone lethality. Taken together, these results demonstrated that knockdown of RFC1 enhances lethality of b‐lapachone and could serve as an effective treatment strategy in cancer conditions.Support or Funding InformationThis work was funded by NIH/NCI 5 R01 CA102792‐16 to DAB.

Proteomic screening reveals the PARylation landscape of base excision repair proteins after Beta‐Lapachone treatment in colorectal cancer cells

Colorectal cancer (CRC) is a leading cause of cancer death and the third most common type of cancer worldwide. In early cases, surgery is the mainstay of treatment, but often patients are primarily diagnosed in an advanced stage of disease and may have distant metastases. Most solid tumors overexpress NAD(P)H;Quinone Oxidoreductase 1 (NQO1), which catalyzes a futile redox cycle of b‐lapachone during exposure, resulting in the formation of high levels of reactive oxygen species (ROS). Futile cycle b‐lapachone recycling produces elevated superoxide levels, resulting in massive levels of hydrogen peroxide that lead to substantial DNA damage and endoplasmic reticulum Ca2+ release. DNA damage and Ca+2 entry into the nucleus leads to the hyperactivation of poly(ADP‐ribose) polymerase 1 (PARP1). A majority of parylation is noted when hyperactivated PARP1 self‐parylates, forming extensive Poly(ADP) ribosylated PAR‐PARP1 levels are noted within 5 mins of exposure to 4 μM β‐lap in A549 cells, as noted by Western blotting using an antibody specific for PARylated moieties. PAR‐PARP1 is a protein post‐translational modification catalyzed by a family of enzymes known as PARPs. DNA single strand breaks along with base‐damage and abasic sites together with nuclear Ca2+ levels lead to hyperactivation of PARP1, a DNA repair enzyme that utilizes NAD+ to generate PAR moieties. This is followed by dramatic NAD+/ATP losses and tumor cells die of a special programmed necrosis process, referred to as NAD+‐keresis.PAR‐PARP1 is rapidly degraded by poly(ADP‐ribose) glycohydrolase (PARG) a exo‐ and endoglycosidase. PARG‐knockdown HCT116 cells were used to gain insight into the function of PARG and whether PARG depletion would enhance b‐lapachone induced cell death by blocking PAR recycling. Proteomic screening after b‐lapachone treatment in colorectal cancer cell line revealed the PARylation landscape of base excision, mismatch and nucleotide excision repair proteins. Twenty (20) of the proteins were found to overlap with H2O2 treatments (200 μM or 2 mM), while one protein (i.e., SPSF) was unique to b‐lapachone treatment. b‐Lapachone treatment affects BER pathways and qualitative analysis of the human Asp‐ and Glu‐ADP‐ribosylated proteome after b‐lapachone treatment indicate major alterations in BER pathways. We confirmed that b‐lapachone mediated cytotoxicity was independent of PARG knockdown or TOPO I or II protein losses. In contrast, RFC1 and PCNA siRNA‐mediated protein knock downs significantly enhanced b‐lapachone lethality, whereas knockdown of Topo I or Topo IIalpha did not affect b‐lapachone lethality. Taken together, these results demonstrated that knockdown of RFC1 enhances lethality of b‐lapachone and could serve as an effective treatment strategy in CRC. We are currently delineating the mechanism of enhanced lethality of β‐lapachone by loss of RFC1.Support or Funding InformationThis work was funded by NIH/NCI 5 R01 CA102792‐16 to DAB.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P53, MAPK, topoisomerase II alpha and Ki67 immunohistochemical expression and KRAS/BRAF mutation in ovarian serous carcinomas

BackgroundWe investigated the immunohistochemical expression of p53, MAPK, topoisomerase II alpha (topoII alpha) and Ki67 in ovarian serous carcinomas (OSCs) along with mutational analysis for KRAS and BRAF.MethodsEighty one cases of OSCs were reviewed and examined immunohistochemically using antibodies against p53, MAPK, topoII alpha and Ki67. Staining was evaluated as a percentage of immunopositive cells with cut-off levels at 10% for p53 and topoII alpha, and 5% for MAPK. The Ki67 immunoexpression was assessed by means of Olympus Image Analysis System as a percentage of immunopositive cells in 1000 tumor cells. KRAS and BRAF mutational analysis was performed on 73 available microdissected samples.ResultsOf 81 cases of OSCs 13.6% were of low-grade and 86.4% were of high-grade morphology. In the high-grade group there was a significantly higher immunoexpression of p53 (P < 0.001) and topoII alpha (P = 0.001), with Ki67 median 56.5 vs. 19 in low-grade group (P < 0.001). The difference in immunoexpression of active MAPK between low- and high-grade group was also significant (P = 0.003). MAPK positive immunostaining was detected in 63.6% of low-grade vs. 17.1% of high-grade OSCs. The frequency of KRAS mutation was significantly higher in low-grade as compared to high-grade group (P = 0.006). None of the samples had BRAF mutation. In addition, we detected positive MAPK immunoexpression in 13/59 samples with wild-type KRAS, suggesting that activation of MAPK pathway is not ultimately related either to KRAS or BRAF mutation. Seven morphologically high-grade samples (11.7%) showed both KRAS mutation and p53 immunopositivity.ConclusionsAlthough this study is limited by its humble number of low-grade samples, our data fit the proposed dualistic pathway of ovarian carcinogenesis. Mutational analysis for KRAS and BRAF discloses some possible interactions between different tumorigenic pathways of low- and high-grade carcinomas. Immunohistochemical staining for MAPK was not sufficiently sensitive, nor specific, to precisely predict the KRAS mutation. However, it appears to be quite reliable in ruling out a KRAS mutation if the staining is negative.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9283563368804632ZusammenfassungHintergrundWir untersuchten die Immunohistochemische Expression der p53, MAPK, topoisomerase II alpha (topoII alpha) und Ki67 in Ovarialkarzinomen (OSCs) anbei mit Mutationsanalyse für KRAS und BRAF.Methode81 OSCs Fälle wurden analysiert und Immunohistochemisch untersucht mit Antikörper gegen p53, MAPK, topoII alpha und Ki67. Die Färbung war ausgewertet als der Prozent von immunopositiven Zellen mit den “cut-of” Niveau an 10% für p53 und topoII alpha und 5% für MAPK. Die Ki67 Expression war bewertet mittels Olympus Image Analysis System als der Prozent von immunopositiven Zellen in 1000 Tumorzellen. KRAS and BRAF Mutationsanalyse wurde in 73 verfügbaren microdissections Stichproben aufgeführt.ErgebnisseVon 81 OSCs Fälle 13.6% zeigte “low-grade” und 86.4% “high-grade” Morphologie. In der “high-grade” Gruppe war eine statistisch bedeutende höhere Expression von p53 (P < 0.001) und topoII alpha (P = 0.001) mit Ki67 median von 56.5 im Gegensatz zu 19 in der “low-grade” Gruppe (P < 0.001). Die Differenz in Immunoexpression von aktiver MAPK zwischen der “low-grade” und “high-grade” Gruppe war statistisch bedeutend (P = 0.003). MAPK positive Expression war in 63.6% der “low-grade” im Gegensatz von 17.1% der “high-grade” Karzinoms bemerkt. Die Häufigkeit der KRAS Mutation war bedeutend höher in “low-grade” im Verglich zu der “high-grade” Gruppe (P = 0.006). Keiner der Stichproben hate BRAF Mutation. Wir haben auch eine positive MAPK Expression in 13/59 der Stichproben mit “wild-type” KRAS bemerkt, was sugeriert das die Aktivation des MAPK Pfads ist nicht letztmalig mit KRAS oder BRAF verbunden. Sieben der “high-grade” Stichproben (11.7%) waren KRAS Mutation und p53 Expression positive.SchlussworteObwohl diese Studie mit bescheiden Nummer von “low-grade” Stichproben limitiert ist, unsere Daten passen in das dualistische Modell von Ovarial Karzinogenesis. Mutationsanalyse für KRAS und BRAF enthüllen einige mögliche Interaktionen zwischen verschieden tumorigenen Wege von “low”- and “high-grade” Karcinomen.Die Immunohistochemische Expression für MAPK war nicht empfindlich oder spezifisch genüg um den KRAS mutations Status des Tumor genau vorauszusagen.Es scheint das die MAPK Expression ziemlich verlässlich ist in ausschließen der KRAS Mutation, wenn die Expression negative ist.

Etoposide-mediated glioblastoma cell death: dependent or independent on the expression of its target, topoisomerase II alpha?

Treatments which significantly improve progression-free and overall survival for patients with relapsed glioblastoma (GBM) after the standard therapy are lacking. The Topoisomerase II (TopoII) enzyme is a key target of anticancer agents because of the important role it plays in transcription regulation and chromatin remodeling. A drug with strong topoisomerase-mediated anticancer activity is etoposide that is used in combination with carboplatin in patients with relapsed GBM. We hypothesized that tumors harboring high expression of TopoII alpha (TopoIIa) would be more sensitive to etoposide treatment. The relative expression levels of TopoIIa protein were measured in a panel of GBM cell lines using Western blot analysis and in a cohort of GBM using immunohistochemistry. Expression levels of TopoIIa in the cell lines were correlated with relative sensitivity to treatment with etoposide. To ascertain the role TopoIIa plays in mediating response to etoposide, expression was reduced with a siRNA targeted to TopoIIa. Protein expression of TopoIIa, although high in the cell lines, was very low in patient specimens. Correlations between TopoIIa protein expression and sensitivity to etoposide were evident. The IC(50) for the low-TopoIIa-expressing cell line, T98G, was almost 50 times higher than M059K (high TopoIIa). Inhibition of TopoIIa in MO59K cells with siRNA significantly altered the IC(50), increasing the resistance to etoposide. Interestingly, the expression of TopoIIa was not decreased after treatment with etoposide, indicating other mechanisms underplay treatment response. In vitro, the levels of TopoIIa protein expression correlate with response to etoposide but also multiple molecular events namely DNA-PK and MDR also play a role in cell sensitivity to etoposide. That we did not find a high expression of TopoIIa in clinical specimens further suggests the mechanisms underlying treatment response are complex.

食道癌におけるTopoisomerase-ⅠおよびTopoisomerase-Ⅱα の発現の検討

Human DNA topoisomerases I and IIalpha (Topo-I and -II alpha) are essential for vital cellular processes such as DNA replication, transcription, translation, recombination, and repair. The purpose of this study was to investigate the clinical significance of the expression of Topo-I and Topo-II alpha. Twenty-nine specimens of esophageal squamous cell carcinoma from patients who had been treated by complete resection of the esophageal tumor were studied by an immunohistochemical analysis. High expression of Topo I and II alpha was identified in 48.7% and 55.2% of tumors, respectively. Neither the Topo-I nor -II alpha expression level had any association with clinical characteristics, including differentiation and the depth of tumor invasion, lymph node metastasis, or the patient prognosis. However, a significant positive correlation was observed between the expression levels of Topo-I and Topo-II alpha. Our study results underscore the potential role of topoisomerase expression in esophageal cancer and further exploratory investigation is necessary to evaluate topoisomerase expression as a surrogate marker in chemotherapy with topoisomerase inhibitor for esophageal cancer.

Topoisomerase IIα expression in acute myeloid leukaemia cells that survive after exposure to daunorubicin or ara-C

Patients diagnosed with acute myeloid leukaemia are often treated with a combination of daunorubicin and 1-beta-D-arabinofuranosylcytosine (ara-C). Both daunorubicin and ara-C exert their effects in the cell nucleus but by different mechanisms, i.e. daunorubicin causes double stranded DNA breaks by inhibition of the nuclear enzyme, topoisomerase (topo) IIalpha, whereas ara-C is an anti-metabolite that integrates with DNA during DNA synthesis and causes cell cycle arrest. Despite the initial efficacy of these drugs, resistance often develops in the clinical setting. The mechanisms underlying clinical resistance to these drugs are poorly understood, but may be associated with an increase in the proportion of topo IIalpha negative cells. Therefore, the aim of this study was to determine whether daunorubicin treatment results in increased numbers of topo IIalpha negative subpopulations in vitro. Acute myeloid leukaemia cells isolated from 12 consenting patients were treated for 24 h with increasing concentrations of daunorubicin or ara-C and the proportion of topo IIalpha-negative cells in surviving cell populations determined by flow cytometry. Treatment with daunorubicin, but not ara-C, resulted in a significant increase in the proportion of topo IIalpha negative cells (p=0.0023). These results suggest that daunorubicin may act by cell cycle arrest and/or by selection of pre-existing topo IIalpha negative subpopulations. Both of these mechanisms can theoretically contribute to a reduced efficacy of a second dose of daunorubicin. The clinical relevance of these interactions should be further elucidated in experimental and clinical studies.

Significance of cell proliferation markers (Minichromosome maintenance protein 7, topoisomerase IIα and Ki‐67) in cavital fluid cytology: Can we differentiate reactive mesothelial cells from malignant cells?

The aim of this study was to evaluate whether immunocytochemical expressions of proliferation markers, such as minichromosome maintenance protein 7 (MCM 7), topoisomerase IIalpha (topo IIalpha), and Ki-67, in reactive mesothelial cells and malignant cells obtained from cavital fluids could be useful for their differential diagnosis. Samples diagnosed as reactive mesothelial cells (14 cases) or malignant tumors (28 cases) in cavital fluids were examined. Immunocytochemical staining of MCM 7, topo IIalpha, and Ki-67 was performed with the universal immunoperoxidase polymer method. In reactive mesothelial cells, MCM 7 was stained in a fine granular pattern and its distribution was uniform in the nuclei. Topo IIalpha and Ki-67 were stained in a coarse granular pattern and the distributions were the same as MCM 7. In contrast, in malignant cells, MCM 7 was stained in an irregular and fine granular pattern, and topo IIalpha and Ki-67 were stained in a uniform and coarse granular pattern. Labeling indices of MCM 7 (cut-off value; 30%, sensitivity; 100%, and specificity; 100%), topo IIalpha (cut-off value; 15%, sensitivity; 89.3%, and specificity; 92.9%) and Ki-67 (cut-off value; 30%, sensitivity; 64.3%, and specificity; 92.9%) of malignant cells were significantly higher than those of reactive mesothelial cells. MCM 7, topo IIalpha, and Ki-67 are different types of cell proliferation markers. MCM 7 and topo IIalpha, in particular, could be reliable tools for differential diagnosis between reactive mesothelial cells and malignant cells.

Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition.

Topoisomerase IIalpha (topo IIalpha) is exported from the nucleus of human myeloma cells by a CRM1-dependent mechanism at cellular densities similar to those found in patient bone marrow. When topo IIalpha is trafficked to the cytoplasm, it is not in contact with the DNA; thus, topo IIalpha inhibitors are unable to induce DNA-cleavable complexes and cell death. Using a CRM1 inhibitor or a CRM1-specific small interfering RNA (siRNA), we were able to block nuclear export of topo IIalpha as shown by immunofluorescence microscopy. Human myeloma cell lines and patient myeloma cells isolated from bone marrow were treated with a CRM1 inhibitor or CRM1-specific siRNA and exposed to doxorubicin or etoposide at high cell densities. CRM1-treated cell lines or myeloma patient cells were 4-fold more sensitive to topo II poisons as determined by an activated caspase assay. Normal cells were not significantly affected by CRM1-topo II inhibitor combination treatment. Cell death was correlated with increased DNA double-strand breaks as shown by the comet assay. Band depletion assays of CRM1 inhibitor-exposed myeloma cells showed increased topo IIalpha covalently bound to DNA. Topo IIalpha knockdown by a topo IIalpha-specific siRNA abrogated the CRM1-topo II therapy synergistic effect. These results suggest that blocking topo IIalpha nuclear export sensitizes myeloma cells to topo II inhibitors. This method of sensitizing myeloma cells suggests a new therapeutic approach to multiple myeloma.

Genistein Inhibition of Topoisomerase IIα Expression Participated by Sp1 and Sp3 in HeLa Cell

Genistein (4′, 5, 7-trihydroxyisoflavone) is an isoflavone compound obtained from plants that has potential applications in cancer therapy. However, the molecular mechanism of the action of genistein on cancer cell apoptosis is not well known. In this study, we investigated the effect of genistein on topoisomerase II-α (Topo IIα), an important protein involved in the processes of DNA replication and cell proliferation. The results revealed that inhibition of Topo IIα expression through the regulation of Specificity protein 1 and Specificity protein 3 may be one of the reasons for genistein’s induction of HeLa cell apoptosis.

Metnase Mediates Resistance to Topoisomerase II Inhibitors in Breast Cancer Cells

DNA replication produces tangled, or catenated, chromatids, that must be decatenated prior to mitosis or catastrophic genomic damage will occur. Topoisomerase IIα (Topo IIα) is the primary decatenating enzyme. Cells monitor catenation status and activate decatenation checkpoints when decatenation is incomplete, which occurs when Topo IIα is inhibited by chemotherapy agents such as the anthracyclines and epididophyllotoxins. We recently demonstrated that the DNA repair component Metnase (also called SETMAR) enhances Topo IIα-mediated decatenation, and hypothesized that Metnase could mediate resistance to Topo IIα inhibitors. Here we show that Metnase interacts with Topo IIα in breast cancer cells, and that reducing Metnase expression significantly increases metaphase decatenation checkpoint arrest. Repression of Metnase sensitizes breast cancer cells to Topo IIα inhibitors, and directly blocks the inhibitory effect of the anthracycline adriamycin on Topo IIα-mediated decatenation in vitro. Thus, Metnase may mediate resistance to Topo IIα inhibitors, and could be a biomarker for clinical sensitivity to anthracyclines. Metnase could also become an important target for combination chemotherapy with current Topo IIα inhibitors, specifically in anthracycline-resistant breast cancer.

A study of proliferative markers and tumor suppressor gene proteins in different grades of ependymomas

Ependymomas are CNS tumors that originate from the spinal canal and walls of the ventricular system. Considerable controversy continues to exist with regard to their prognostic factors; age and extent of resection are the only statistically significant prognostic factors yet identified. The authors report a retrospective study of a homogenous population of 119 patients harbouring ependymomas between 1991 and 2002. All clinico-radiological and follow-up data were analyzed and a pathologic review was performed by two pathologists. Immunohistochemical staining for MIB-1, Topo IIalpha, p53 and MDM2 was performed. Histopathologic grades show relationship with MIB1 and Topo IIalpha labelling indices and cut-off values of 5% can differentiate between anaplastic and lower grades. p53 and MDM2 proteins expression are not common in ependymomas; however, they are seen in higher grades only and may be involved in the tumor progression.

HMGB1 and HMGB2 proteins up-regulate cellular expression of human topoisomerase II

Topoisomerase IIα (topo IIα) is a nuclear enzyme involved in several critical processes, including chromosome replication, segregation and recombination. Previously we have shown that chromosomal protein HMGB1 interacts with topo IIα, and stimulates its catalytic activity. Here we show the effect of HMGB1 on the activity of the human topo IIα gene promoter in different cell lines. We demonstrate that HMGB1, but not a mutant of HMGB1 incapable of DNA bending, up-regulates the activity of the topo IIα promoter in human cells that lack functional retinoblastoma protein pRb. Transient over-expression of pRb in pRb-negative Saos-2 cells inhibits the ability of HMGB1 to activate the topo IIα promoter. The involvement of HMGB1 and its close relative, HMGB2, in modulation of activity of the topo IIα gene is further supported by knock-down of HMGB1/2, as evidenced by significantly decreased levels of topo IIα mRNA and protein. Our experiments suggest a mechanism of up-regulation of cellular expression of topo IIα by HMGB1/2 in pRb-negative cells by modulation of binding of transcription factor NF-Y to the topo IIα promoter, and the results are discussed in the framework of previously observed pRb-inactivation, and increased levels of HMGB1/2 and topo IIα in tumors.

Prognostic significance of β‐catenin and topoisomerase IIα in de novo acute myeloid leukemia

The Wnt/beta-catenin signaling is important for controlling self-renewal of hematopoietic stem cells and its constitutive activation has recently been documented in a significant proportion of acute myeloid leukemia (AML) cases. Topoisomerase IIalpha (Topo IIalpha) is a marker of cell proliferation and a crucial target for anthracycline cytotoxicity, the mainstay of management employed in AML. We retrospectively investigated the prognostic roles of beta-catenin and topo IIalpha in a cohort of 59 patients with newly diagnosed AML by immunohistochemistry. Aberrant beta-catenin expression was demonstrated in 13 patients (22%), and it was more likely to occur in those with unfavorable karyotypes. Advanced age and poor performance status adversely influenced the achievement of complete remission, while neither aberrant beta-catenin expression nor enhanced topo IIalpha activity did. On multivariate survival analysis, four factors independently predicted a shortened overall survival: aberrant beta-catenin expression, high topo IIalpha activity, poor-risk cytogenetics, and presence of at least one comorbidity factor. Our results suggest that both beta-catenin and topo IIalpha independently predicted an adverse prognosis and might serve as new markers for risk stratification in AML patients.

Analysis of SUMOylation of topoisomerase IIalpha with Xenopus egg extracts.

Posttranslational protein modification by the Small Ubiquitin-like MOdifiers (SUMO) is involved in many cellular functions including organization of nuclear structures and chromatin, transcriptional regulation, and nucleo-cytoplasmic transport. Both genetic and biochemical studies indicate that the SUMO modification pathway plays an important role in proper cell cycle control, especially in the normal progression of mitosis. DNA topoisomerase II has been shown to be modified by SUMO in budding yeast as well as in vertebrates. We have shown by biochemical analysis using the Xenopus egg extract (XEE) cell-free assay system that DNA topoisomerase IIalpha (Topo IIalpha) is modified by SUMO-2/3 on mitotic chromosomes in the early stages of mitosis. Inhibition of mitotic SUMOylation in the XEE assay system causes aberrant sister chromatid separation in anaphase and alters Topo IIalpha association with chromosomes.

Topoisomerase IIα controls the decatenation checkpoint

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.

Expression of Human DNA Topoisomerase II-α in Squamous Cell Carcinoma of the Larynx and Its Correlation With Clinicopathologic Variables

The aggressiveness of laryngeal squamous cell carcinoma (SCC) is unpredictable. Topoisomerase (Topo) II-alpha is an essential nuclear enzyme; its expression rises at the end of the S-G2/M phase and drops at completion of mitosis. This study sought to determine if Topo II-alpha expression can serve as a prognostic factor in laryngeal SCC. Specimens from 56 consecutive patients were immunohistochemically stained for Topo II-alpha, and the number of positive cells in the areas of highest staining was counted in 3 highpower fields (X400) (Topo II-alpha index). Differences in the Topo II-alpha index by the presence or absence of recurrence, tumor stage and grade, and disease course were analyzed statistically. On multivariate Cox regression analysis, the Topo II-alpha index (>70 or < or =70) (P = .008) and tumor grade (P = .034) independently predicted disease-free survival. These findings suggest that high Topo II-alpha expression may be a useful indicator of tumor aggressiveness and poor outcome in laryngeal SCC.

Casein kinase I / phosphorylates topoisomerase II at serine-1106 and modulates DNA cleavage activity

We previously reported that phosphorylation of topoisomerase (topo) IIα at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Iδ and/or CKIɛ, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II–DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II–DNA cleavable complex formation. Since, IC261 specifically targets the Ca2+-regulated isozymes, CKIδ and CKIɛ, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIα and α′ did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIδ/ɛ homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIα, was enhanced following expression of human CKIɛ. Down-regulation of CKIδ and CKIɛ also led to reduced formation of etoposide stabilized topo II–DNA cleavable complex. These results provide strong support for an essential role of CKIδ/ɛ in phosphorylating Ser-1106 in human topo IIα and in regulating enzyme function.

HER2 and topoisomerase IIα: possible predictors of response to neoadjuvant chemotherapy for breast cancer patients

Surrogate markers may be used to assess the response to neoadjuvant treatment. The association between HER2 overexpression and favorable response to specific therapy in breast cancer is controversial, and the mechanism unclear. The purpose of the study was to evaluate HER2 and topoisomerase IIalpha (Topo IIalpha) as candidates for predicting the response to neoadjuvant chemotherapy in breast cancer patients. Between 1999 and 2006, seventy-six breast cancer patients who had received neoadjuvant chemotherapy were studied. Regimens including either CEF (cyclophosphamide, epirubicin, 5-fluorouracil) or CMF (cyclophosphamide, methotrexate, 5-fluorouracil) were given in more than three cycles to this group of patients. Protein expression of HER2 and Topo IIalpha were determined by immunohistochemistry. The primary endpoint was pathological and clinical response. Of 76 primary breast cancer samples, 27 (35.5%) showed overexpression of either HER2 (25%) or Topo IIalpha protein (10.5%), whereas in 7 tumors (9.2%) both proteins were found to be overexpressed. Ten patients (13.2%) had a clinical complete response and 21 (27.6%) had a clinical partial response. Five women (6.6%) had a pathological complete response, 5 (6.6%) had microscopic residual disease, and 46 (60.5%) had macroscopic residual disease. HER2 and Topo IIalpha overexpression was significantly associated with a favorable response (P < 0.001 and P = 0.005 respectively). Our study suggests that HER2 and Topo IIalpha overexpression could be predictors of the response to neoadjuvant chemotherapy in both the CEF and CMF arms.

Paclitaxel-doxorubicin sequence is more effective in breast cancer cells with heat shock protein 27 overexpression

Cancer cells with overexpression of heat shock protein 27 (HSP27) are resistant to chemotherapeutic drug doxorubicin (Dox). Paclitaxel (Pacl) was reported to suppress HSP27 expression in ovarian and uterine cancer cells. The purposes of this study were to investigate whether Pacl inhibits the expression of HSP27 in breast cancer cells, whether Pacl can sensitize breast cancer cells with HSP27 overexpression to Dox, and to define a more effective schedule for the combination of Dox with Pacl. The HSP27 high-expressing human breast cancer cell lines, MCF-7 and MDA-MB-435, and the HSP27 low-expressing cell line, MDA-MB-231, were used in this study. The level of HSP27, topoisomerase (Topo) IIalpha and beta expression were assessed by Western blotting. The cytotoxic activities of Dox, Pacl and combination of these two drugs were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and flow cytometric assays. Pacl (0.1 micromol/L) inhibited HSP27 expression by approximately 2-fold in MCF-7 and MDA-MB-435 cells, while up-regulating the level of topo IIalpha and beta. In contrast, expression of HSP27 in MDA-MB-231 did not change significantly following Pacl treatment. There were synergistic effects in both treatment sequences (Pacl-Dox and Dox-Pacl) when Pacl was combined with Dox. Compared with those treated with the Dox-Pacl sequence, the Pacl-Dox sequence had a stronger effect in cancer cells with HSP27 overexpression, as MCF-7 and MDA-MB-435 treated with the Pacl-Dox sequence had lower viabilities and a higher apoptotic rate. Paclitaxel significantly decreases the level of HSP27 in breast cancer cells overexpressing HSP27. In combination therapies, the Pacl-Dox sequence is more effective in clearing breast cancer cells with high HSP27 expression compared with the Dox-Pacl sequence.