Expression Of Topo Research Articles

Comparison of biomarker expression in oral lichen planus and oral lichenoid lesions.

Oral lichen planus (OLP) and oral lichenoid lesions (OLL) comprise a group of oral mucosal disorders that have similar clinical and histological features. To compare the levels of investigated biomarkers in biopsied OLP and OLL, and to determine the pattern of biomarkers, which could be useful for the biological characterization of these 2 disorders. A total of 56 biopsy specimens in 2 groups were analyzed in this study. One group consisted of 25 idiopathic OLP lesions, and the other included 31 OLL from patients treated with antihypertensive and cardiac medications. The expression of protein p53, topoisomerase I (topo I), heat shock protein 90 (HSP90), and E-cadherin was analyzed using immunohistochemistry. The p53 protein expression showed a trend to a positive correlation with topo I expression in the total sample (p = 0.067, R = 0.25). The p53 protein and HSP90 expression was higher in the OLL group compared to the OLP group, but the difference was not statistically significant. No association was found between topo I and E-cadherin expression for either the OLP or OLL group. The findings of this study suggest that the slightly higher protein p53 and HSP90 expression in the OLL group might be caused by the medications used. The slight association between p53 and topo I expression indicates that the cooperation between these proteins might be essential for the growth of OLP/OLL in general. We conclude that the overexpression of p53 protein and high expression of topo I found in both types of lesions might induce their biologically aggressive behavior.

New Benzochromeno[2,3-d]Pyrimidines and Benzochromenotriazolo[1,5-c]Pyrimidines as Potential Inhibitors of Topoisomerase II

A group of novel benzochromenopyrimidine and benzochromenotriazolopyrimidine derivatives was designed and synthesized by a series of distinct reactions, beginning with the ethyl formimidate derivative 2, and evaluated as selective inhibitors of Topoisomerase II. Hydrazinolysis of compound 2 yielded the corresponding amino–imino 7, which in turn was exploited to produce the target benzochromenotriazolopyrimidine derivatives. The chemical structures of target derivatives were assured by different elemental and spectral analyses. The antineoplastic activity of the new benzochromene derivatives against human liver (HepG2), breast (MCF7), and colon (HCT-29) cancer cells was investigated and compared to doxorubicin. Derivatives 3, 8, 11, 12, and 13 had significant (<10 µM) antiproliferative activities. With the exception of derivative 13, the other efficient anticancer agents were selective and did not affect the growth of the normal fibroblasts (WI-38). Derivatives 8, 11, and 12 elevated the expression of p53 and caspase 3, threefold–fivefold. Derivative 3 induced the expression of caspase 3 without affecting that of p53. Derivative 11 was unique in inhibiting the expression of cdk1 by approximately 60%. All derivatives (3, 8, 11, and 12) reduced the expression of topo II by 46–56%. All these derivatives induced apoptosis as indicated by the elevation (∼fourfold–fivefold) in the activity of the cleaved active caspase 3. Derivatives 8, 11, and 12 reduced the concentration of topoisomerase II protein by approximately 50%. The antiproliferative activity of the new benzochromene derivatives is partially caused by their ability to downregulate the topoisomerase II and induce apoptosis.

Association of immunophenotype with expression of topoisomerase II \u03b1 and \u03b2 in adult acute myeloid leukemia

Anthracyclines used in the treatment of acute myelogenous leukemia (AML) inhibit the activity of the mammalian topoisomerase II (topo II) isoforms, topo II α and topo IIβ. In 230 patients with non-M3 AML who received frontline ara-C/daunorubicin we determined expression of topo IIα and topo IIβ by RT-PCR and its relationship to immunophenotype (IP) and outcomes. Treatment outcomes were analyzed by logistic or Cox regression. In 211 patients, available for analysis, topo IIα expression was significantly lower than topo IIβ (P < 0.0001). In contrast to topo IIα, topo IIβ was significantly associated with blast percentage in marrow or blood (P = 0.0001), CD7 (P = 0.01), CD14 (P < 0.0001) and CD54 (P < 0.0001). Event free survival was worse for CD56-negative compared to CD56-high (HR = 1.9, 95% CI [1.0–3.5], p = 0.04), and overall survival was worse for CD-15 low as compared to CD15-high (HR = 2.2, 95% CI [1.1–4.2], p = 0.02). Ingenuity pathway analysis indicated topo IIβ and immunophenotype markers in a network associated with cell-to-cell signaling, hematological system development/function and inflammatory response. Topo IIβ expression reflects disease biology of highly proliferative disease and distinct IP but does not appear to be an independent variable influencing outcome in adult AML patients treated with anthracycline-based therapy.

Drug resistance gene expression and chemotherapy sensitivity detection in Chinese women with different molecular subtypes of breast cancer.

Objective:The aim of the study was to identify specific chemosensitivity drugs for various molecular subtypes of breast tumors in Chinese women, by detecting the expression of drug resistance genes and by using the drug sensitivity test on different molecular subtypes of breast cancers.Methods:The expression of drug resistance genes including Topo II, GST-π, P-gp, LRP, and CD133 were detected with immunohistochemistry in a tissue microarray. Drug sensitivity tests included those for paclitaxel, epirubicin, carboplatin, vinorelbine, and fluorouracil and were conducted on primary cancer tissue cells and cell lines, including the T47D, BT-474, and MDA-MB-231 cells and human breast cancer xenografts in nude mice.Results:The different drug resistant genes Topo II, GST-π, P-gp, and LRP were differentially expressed among different molecular subtypes of breast cancers (P < 0.05). Positive expression of CD133 was highest in basal-like breast cancer (P < 0.05). Kaplan-Meier survival analysis showed that positive expressions of Topo II and CD133 both correlated with shorter disease-free survival (DFS) (P < 0.05) and overall survival (P < 0.05), and positive expression of LRP correlated only with shorter DFS (P < 0.05). BT-474 showed chemosensitivity to paclitaxel and epirubicin, while MDA-MB-231 showed chemosensitivities to paclitaxel, epirubicin, carboplatin, and fluorouracil (T/C ≤ 50%). The basal-like and HER2+ breast cancer primary cells showed chemosensitivities to paclitaxel and epirubicin with significant differences compared with luminal breast cancer primary cells (P < 0.05).Conclusions:The differential expression of drug resistance genes and the differential chemosensitivities of drugs in different molecular subtype of breast cancers suggested that individual treatment should be given for each type of breast cancer.

Design, molecular modeling and anticancer evaluation of thieno[2,3-d]pyrimidine derivatives as inhibitors of topoisomerase II.

Synthesis of 4-(3,5-dimethyl-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine 1 and its functionalized reactions as nucleophile with various electrophilic reagents were performed through facile methods to yield different cyclic and acyclic derivatives (2-17). The structures of the newly synthesized compounds were established by their elemental analysis and spectral data. Derivatives 4, 14, 16, and 17 in addition to the parent compound 1 had IC50 at ~4-10µM against HepG2 and MCF7 and were selected for further investigations. All derivatives had high IC50 values (>60µM) against normal fibroblasts WI38 indicating selectivity against cancer cell lines. Derivatives 4, 14, and 17 up-regulated the expression of p53 by ~3-4 folds. All derivatives caused a significant ~3-fold increase in the expression of executive caspase 3 and significant elevation in cleaved caspase 3 activity. The elevation in the expression of caspase 3 by compound 1 and derivative 16 was not accompanied by an increase in p53 expression or cleaved caspase 3 activity. These two thienopyrimidines may act directly on caspase 3. Derivative 17 was unique in reducing the expression of Topo II by ~60%. The molecular docking showed that derivatives 4 and 17 with high binding energies could bind and inhibit Topoisomerase II (Topo II). In accordance with the docking modelling, derivatives 4 and 17 reduced the Topo II concentration by 82 and 90%, respectively, compared to the untreated cells. However, the parent compound 1 also caused a significant 34% reduction in the enzyme concentration although it was not predicted as a ligand for the enzyme in the docking study. Taken together, derivatives 4, 14 and 17 showed selective cytotoxicity, could arrest cell cycle, and induce apoptosis. Furthermore, they could serve as cytostatic agents by inhibiting/reducing Topo II.

Impact of the colistin resistance gene mcr-1 on bacterial fitness

A Klebsiella pneumoniae isolate harbouring a 217 kb IncHI2-type plasmid (pKP2442) encoding the colistin resistance gene mcr-1 was isolated from a leukaemia patient. pKP2442 was mobilised by intragenus and intergenus transconjugation from the clinical isolate to Escherichia coli J53 (transconjugation frequency 6.86 × 10−8 ± 5.57 × 10−8) and K. pneumoniae PRZ (transconjugation frequency 4.04 × 10−8 ± 3.03 × 10−8), respectively. Since acquisition of resistance determinants often results in a loss of fitness, the impact of mcr-1 on the fitness of E. coli and K. pneumoniae was investigated. Escherichia coli J53 and K. pneumoniae PRZ transformants harbouring the TOPO expression vector encoding mcr-1 displayed significantly decreased growth rates compared with isogenic parental strains and controls. In contrast, competitive growth experiments revealed equal growth rates between E. coli J53 pKP2442 transconjugants (TcpKP2442) and the parental strain, whereas K. pneumoniae PRZ TcpKP2442 showed significantly reduced growth rates compared with their parental strain (selection rate constant −1.62 ± 0.49), indicating a decrease in fitness. Infection of A549 human lung epithelial cells with TcpKP2442 or mcr-1 transformants and controls revealed equal lactate dehydrogenase activities, indicating no significant impact of mcr-1 on cytotoxicity. Likewise, survival of Galleria mellonella larvae infected with mcr-1-expressing strains and isogenic controls was similar. These data indicate that expression of mcr-1 is able to cause a fitness cost when encoded on expression vectors and that acquisition of natural plasmid-borne mcr-1 does not impair fitness in E. coli J53 but negatively influences growth rates in K. pneumoniae PRZ.

Heat Resistance of Histidine Decarboxylase from Gram-Negative Histamine-Producing Bacteria in Seafood

Precooking of tuna is a potential critical control point (CCP) in the commercial manufacturing of canned tuna. To assess the efficacy of precooking as a CCP, an understanding of the thermal properties of histamine-producing bacteria (HPB) and their histidine decarboxylase (HDC) enzymes is required. The thermal properties of many HPB have been determined, but the thermal resistances of the HDC enzymes are unknown. The purpose of this study was to determine the D- and z-values of selected HDC enzymes to evaluate the CCP of precooking during the canning process and provide scientific data to support U.S. Food and Drug Administration guidelines. HDC (hdc) genes from three strains each of Morganella morganii, Enterobacter aerogenes, Raoultella planticola, and Photobacterium damselae were cloned, expressed, and purified using the Champion pET Directional TOPO Expression System, pET100 cloning vector, and HisPur Cobalt resin. The heat resistances of all enzymes were compared at 50°C, and the D- and z-values from one strain of each HPB were determined at 50 to 60°C. To evaluate the heat inactivation of HDC enzymes during canned tuna processing, tuna tissue was inoculated with HDCs and heated to 60°C in a water bath set at 65 and 100°C. The D-values for the HDC enzymes from M. morganii, E. aerogenes, R. planticola, and P. damselae ranged from 1.6 to 4.1, 1.6 to 6.3, 1.9 to 4.3, and 1.6 to 2.9 min, respectively, at 50 to 60°C. The z-values for M. morganii, E. aerogenes, R. planticola, and P. damselae were 19.2, 18.0, 22.0, and 13.3°C, respectively. The HDCs from all HPB except E. aerogenes showed no significant activity after being heated to 60°C. The data generated in this study will help refine current guidelines for the thermal destruction of the HDC enzymes.

Anti-Proliferative and Apoptosis-Inducing Effect of Theabrownin against Non-small Cell Lung Adenocarcinoma A549 Cells.

With the highest cancer incidence rate, lung cancer, especially non-small cell lung cancer (NSCLC), is the leading cause of cancer death in the world. Tea (leaves of Camellia sinensis) has been widely used as a traditional beverage beneficial to human health, including anti-NSCLC activity. Theabrownin (TB) is one major kind of tea pigment responsible for the beneficial effects of tea liquor. However, its effect on NSCLC is unknown. The aim of the present study was to evaluate anti-proliferative and apoptosis-inducing effect of TB on NSCLC (A549) cells, using MTT assay, morphological observation (DAPI staining), in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and annexin-V/PI flow cytometry. Subsequently, the expression of several genes associated with cell proliferation and apoptosis were detected by real time PCR assay to explore its potential underlying mechanism. TB was revealed to inhibit cell proliferation of A549 cells in a concentration-dependent and time-dependent manner. Morphological observation, TUNEL assay and flow cytometric analysis evidenced an apoptosis-inducing effect of TB on A549 cells in a concentration-dependent manner. The real time PCR assay demonstrated that TB down-regulated the expression of TOPO I, TOPO II, and BCL-2, and up-regulated the expression of E2F1, P53, GADD45, BAX, BIM, and CASP 3,7,8,9, which suggests an activation of P53-mediated apoptotic (caspase-dependent) pathway in response to TB treatment. The western blot analysis showed a similar trend for the corresponding protein expression (P53, Bax, Bcl-2, caspase 3,9, and PARP) and further revealed DNA damage as a trigger of the apoptosis (phosphorylation of histone H2A.X). Accordingly, TB can be speculated as a DNA damage inducer and topoisomerase (Topo I and Topo II) inhibitor that can up-regulate P53 expression and subsequently modulate the expression of the downstream genes to induce cell proliferation inhibition and apoptosis of A549 cells. Our results indicate that TB exhibits its anti-NSCLC activity via a P53-dependent mechanism, which may be a promising candidate of natural product for anti-cancer drug development in the treatment of NSCLC.

Expression of excision repair cross-complementing 1, topoisomeraseII, ribonucleotide reductase M1, β3-tubulin and thymidylate synthase in lung cancer

Objective To analyze the expression characteristics of excision repair cross-complementing 1(ERCC1), topoisomerase Ⅱ (TOPO Ⅱ), ribonucleotide reductase M1(RRM1), β3-tubulin and thymidylate synthase (TS) in lung cancer and their associations with the pathological types. Methods The immunohistochemical EnVision method was used to determine the expression of ERCC1, TOPOⅡ, RRM1, β3-tubulin and TS in 548 patients who were diagnosed as lung cancer from January 2011 to December 2014. Variance analysis was performed to analyze their expression characteristics among different pathological types and correlation. Results The expression positive rates of ERCC1, TOPOⅡ, RRM1, β3-tubulin and TS were 61.86% (339/548), 91.06% (499/548), 62.59% (343/548), 73.18% (401/548) and 70.44% (386/548), respectively. The expression of ERCC1 was weak positive mostly (P<0.05), meanwhile the expression of TOPO Ⅱ was medium-strong positive mostly (P<0.05). In ERCC1 group, the positive rate of squamous cell carcinoma was higher than that of adenocarcinoma [57.39% (167/291) vs. 42.61% (124/291), P=0.000]. In weak positive of TOPOⅡ group, the proportion of adenocarcinoma was higher than that of squamous cell carcinoma[23.58% (100/137) vs. 8.73% (37/137), P=0.000]. In medium-strong positive of TOPO Ⅱ group, the proportion of squamous cell carcinoma was higher than that of adenocarcinoma[47.41% (201/287/) vs. 20.28% (86/287), P=0.000]. The expressions of ERCC1, TOPO Ⅱ, RRM1, β3-tubulin and TS were irrelevant (r=0.4, P=0.397). Conclusions The expressions of ERCC1 and TOPO Ⅱ are higher in squamous cell carcinoma than those in adenocarcinoma. The expression of ERCC1 is weak positive mostly, meanwhile the expression of TOPO Ⅱ is medium-strong positive mostly. There is no correlation between them. Key words: Lung neoplasms; Excision repair cross-complementing 1; Topoisomerase Ⅱ; Thymidylate synthase

Role in proinflammatory response of YghJ, a secreted metalloprotease from neonatal septicemic Escherichia coli

Neonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a systemic inflammatory response with production and release of a wide range of inflammatory mediators. The increased serum levels of cytokines were found to correlate with the severity and mortality in course of sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflammatory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatal septicemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant by successive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showed homology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ was found to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptide substrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7–8 and at temperatures 37–40°C. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJ stimulated the secretion of cytokines IL-1α, IL-1β and TNF-α in murine macrophages (RAW 264.7) and IL-8 from human intestinal epithelial cells (HT-29). YghJ also down-regulated the production of anti-inflammatory cytokines such as IL-10. YghJ is present in both septicemic (78%) and fecal E. coli isolates (54%). However, expression and secretion of YghJ is significantly higher among the septicemic (89%) than the fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggering proinflammatory response in NSEC.

Topoisomerase I expression is associated with prognosis in postoperative non-small cell lung cancer patients.

BackgroundBiomarkers may help to improve non‐small cell lung cancer (NSCLC) prognosis. However, the prognostic effect of topoisomerase I (Topo I) on NSCLC is unknown. We evaluated the clinicopathologic and prognostic significance of tumor Topo I and thymidylate synthase (TS) protein expression in postoperative NSCLC patients.MethodsOne hundred and fifteen patients with postoperative NSCLC were enrolled. Topo I and TS protein were detected in removed tumors by immunohistochemistry. The correlations between Topo I/TS protein expression and clinicopathologic characters and outcomes of patients were analyzed.ResultsIncreased expression of Topo I was found in 57 (49.6%) tumors. The largest diameter of the tumor was significantly different between patients with high and low Topo I expression (P = 0.035). TS staining showed that 35 (30.4%) carcinomas were TS positive. The level of TS expression was correlated with tumor differentiation (P = 0.037). Patients with low Topo I expression had significantly longer overall survival (OS) than those with high expression (P = 0.004). The correlation between Topo I expression and OS was demonstrated among patients with squamous cell carcinoma (P = 0.030) and patients in pathological tumor node metastasis stage I (P = 0.027). Topo I expression was positively correlated with TS expression in tumor tissue (R = 0.251, P = 0.007).ConclusionsLow Topo I expression is an independent favorable prognostic factor for longer OS in postoperative NSCLC patients, especially in squamous cell carcinoma. There is a correlation between the expression of TS and Topo I in removed tumor tissue.

Mda-7/IL-24 enhances sensitivity of B cell lymphoma to chemotherapy drugs.

Interleukin-24 (IL-24) is a cytokine encoded by a tumor suppressor gene of the IL-10 family, also known as the melanoma differentiation associated gene-7 (Mda-7) and first discovered in human melanoma cells. Mda-7/IL-24 has been shown to inhibit the proliferation of various human tumor cell lines, but its effect on the sensitivity of B cell lymphoma to chemotherapy agents is not yet clear. The present study investigated the effects of Mda-7/IL-24 overexpression on the sensitivity of human B cell lymphoma cells to chemotherapy, as well as its mechanism of action. The sensitivity of stable Mda-7/IL-24 overexpressing Raji and Daudi cells to cis-diamminedichloroplatinum (CDDP), epirubicin and vinblastine (VCR) were assessed by the MTS method, and the IC50 value calculated. Cell apoptosis and the intracellular accumulation of Rhodamine-123 were assayed by flow cytometry. The expression of multidrug resistance gene 1 (MDR1), B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1), topoisomerase II (Topo II) and multidrug resistance-related protein 1 (MRP1) mRNA and protein were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. In addition, western blot analysis was also used to investigate the effect of Mda-7/IL-24 on activity of GTP-RhoA-ERK signaling pathway in Raji and Daudi cells. Growth inhibition and apoptosis rates of Mda-7/IL-24 overexpressing Raji and Daudi cells were higher than those of non-transfected cells and cells transfected with vector alone when treated with CDDP, epirubicin and VCR. The IC50 values of CDDP, epirubicin and VCR were lower for Mda-7/IL-24-overexpressing Raji and Daudi cells than for non-transfected cells and cells transfected with empty vector. Intracellular accumulation of Rhodamine-123 and the expression of Topo II were higher, while the levels of MDR1, BMI and MRP1 mRNA and protein were lower, in Mda-7/IL-24 overexpressing Raji and Daudi cells. Furthermore, the activities of GTP-RhoA-ERK signaling pathway in Raji and Daudi cells were suppressed. These results indicated that Mda-7/IL-24 enhanced the sensitivity of B lymphoma cells to chemotherapy agents by altering the expression of multidrug-resistance genes via downregulating GTP-RhoA-ERK signaling pathway, suggesting that treatment of B cell lymphomas with Mda-7/IL-24 could avoid MDR.

Characterization of Aldolase and Transaldolase Activities in Acidobacterium capsulatum

Acidobacterium capsulatum, a mildly acidophilic, chemotrophic, aerobe with the ability to grow on glucose, is found in diverse soil and aquatic conditions worldwide, suggesting a significant role in carbon and nutrient cycling in the environment. A. capsulatum has successfully been cultured and its genome has been completed, allowing for genome-directed studies of the organism's glucose metabolizing capabilities. Based on the genomic information, this organism was thought to lack aldolase, but contained two putative transaldolase genes (TA), one of which contains and EF-hand domain (TAEF). However, recent results show that there are both aldolase and transaldolase activities in cell-free lysate. We are continuing to characterize the activities of aldolase and transaldolase in cell-free lysate to better understand partitioning of carbon from glucose to energy production and biomass formation. Characterization will include pH and EDTA dependent studies to determine aldolase and transaldolase activity dependence on pH and calcium. Furthermore the two putative transaldolase genes have been amplified, cloned into E. coli and transformed into a pET151 directional TOPO expression vector. Experiments to determine whether the transaldolase genes can be expressed are in progress. Sequencing of the TAEF gene showed proper insertion into the expression vector. Current work is focusing on purification and expression of the TA and TAEF proteins. The evidence from these studies will allow for a better understanding of the role of transaldolase and aldolase as mediators of carbon partitioning between energy production and biomass formation in A. capsulatum. Future work will focus on identifying an aldolase gene and characterizing key regulatory points of glucose metabolism.

β-Elemene Inhibits Cell Proliferation by Regulating the Expression and Activity of Topoisomerases I and IIα in Human Hepatocarcinoma HepG-2 Cells.

Objective. To investigate the effects of β-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells. Methods. After treatment with β-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIα was analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect of β-ELE on DNA breaks. Results. β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration. Conclusion. β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα.

Abstract 786: Alternative RNA processing leads to decreased DNA topoisomerase IIα in etoposide (VP-16) resistant human leukemia K562 cells

Abstract Acquired resistance to the DNA topoisomerase II (topo II)-targeting agent, etoposide (VP-16) has been shown previously in this lab to be associated with decreased levels of topo II mRNA and protein in a K562 clone, K/VP.5. Utilizing exon junctional Taqman hydrolysis probes, real-time quantitative PCR (qPCR) revealed ∼2-fold decrease in topo IIα mRNA expression in K/VP.5 compared to parental K562 cells when probes were downstream of exon 19. However, use of probes upstream of exon 19 revealed a small but statistically significant increase in topo IIα mRNA in K/VP.5 compared to K562 cells. In contrast, expression of topo IIß mRNA was reduced in K/VP.5 compared to K562 cells in all 5’ and 3’ regions of the gene queried. RNA stability studies using the RNA polymerase II inhibitor DRB and 4 qPCR probes, regionally targeted to 3’ and 5’ domains, revealed similar topo IIα mRNA half-lives in K562 cells (6.55 hr [extreme 3’ probe] to 7.96 hr [extreme 5’ probe]) but a statistically significant increase in topo IIα mRNA half-lives in K/VP.5 cells (7.21 hr [extreme 3’ probe] to 11.86 hr [extreme 5’ probe (p=0.034)]). The differential RNA decay rate in K/VP.5 cells suggested alterations in exo- or endo-nuclease expression/function in resistant cells and/or alternative splicing events generating transcripts of different size and stability characteristics. Experiments using Rapid Amplification of cDNA Ends (3’-RACE) were consistent with loss of ∼3 kb of the total 5.8 kb topo IIα in K/VP.5 cells. PCR products derived from this truncated transcript were sequenced, revealing that the 5’ splice site of intron 19 is suppressed, resulting in the retention of ∼300 nt of intronic sequence in the mature mRNA, and the use of an alternative polyadenylation site. The retained portion of intron 19 contains an in-frame stop codon, as well as the consensus AAUAAA hexamer of the poly(A) signal. As a result of alternative 3’ end processing, exons 20-35 of the topo IIα gene are not included in the truncated transcript. Since the codon corresponding to the topo IIα active site tyrosine is contained in Exon 20 (Tyr 705), translation of the alternatively spliced transcript likely produces a non-functional protein. Surprisingly, the truncated transcript is also present in K562 cells, albeit at a greatly reduced level compared to K/VP.5, suggesting that alternative polyadenylation is a normal mechanism to regulate topo IIα expression. Taken together results indicate that posttranscriptional alterations in topo IIα may account, in part, for acquired resistance to VP-16, and potentially for regulation of topo II expression that may dictate intrinsic chemosensitivity as well. Citation Format: Lucas D. Serdar, Ragu Kanagasabai, Jack C. Yalowich. Alternative RNA processing leads to decreased DNA topoisomerase IIα in etoposide (VP-16) resistant human leukemia K562 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 786. doi:10.1158/1538-7445.AM2014-786

DNA topoisomerase IIβ as a molecular switch in neural differentiation of mesenchymal stem cells.

Two isoforms of DNA topoisomerase II (topo II) have been identified in mammalian cells, named topo IIα and topo IIβ. Topo IIα plays an essential role in segregation of daughter chromosomes and thus for cell proliferation in mammalian cells. Unlike its isozyme topo IIα, topo IIβ is greatly expressed upon terminal differentiation of neuronal cells. Although there have been accumulating evidence about the crucial role of topo IIβ in neural development through activation or repression of developmentally regulated genes at late stages of neuronal differentiation, there have been no reports that analyzed the roles of topo IIβ in the neural trans differentiation process of multipotent stem cells. Terminal differentiation of neurons and transdifferentiation of Mesenchymal Stem Cells (MSCs) are two distinct processes. Therefore, the functional significance of topo IIβ may also be different in these differentiation systems. MSC transdifferentiation into neuron-like cells represents an useful model to further validate the role of topo IIβ in neuronal differentiation. The aim of this study is to evaluate the subset of genes that are regulated in neural transdifferentiation of bone marrow-derived human MSCs (BM-hMSCs) in vitro and find genes related with topo IIβ. For this purpose, topo IIβ was silenced by specific small interfering RNAs in hMSCs and cells were induced to differentiate into neuron-like cells. Differentiation and silencing of topo IIβ were monitored by real-time cell analysis and also expressions of topo II isoforms were analyzed. Change in transcription patterns of genes upon topo IIβ silencing was identified by DNA microarray analysis, and apparently genes involved in regulation of several ion channels and transporters, vesicle function, and cell calcium metabolism were particularly affected by topo IIβ silencing suggesting that topoIIβ silencing can significantly alter the gene expression pattern of genes involved in variety of biological processes and signal transduction pathways including transcription, translation, cell trafficking, vesicle function, transport, cell morphology, neuron guidance, growth, polarity, and axonal growth. It appears that the deregulation of these pathways may contribute to clarify the further role of topo IIβ in neural differentiation.

Expressions of cyclooxygenase-2, vascular endothelial growth factor, and topoisomerase II in triplenegative breast cancer and their clinical significance

Objective To research the expression of cyclooxygenase-2 (COX-2),vascular endothelial growth factor (VEGF),and topoisomerase Ⅱ (Topo Ⅱ) and their clinical significance in triple-negative breast cancer (TNBC).Methods We selected 107 cases of breast cancers from our hospital.Tissue chips were prepared with a manufacturing device for tissue microarry.Immunohistochemistry was used to detect COX-2,VEGF,and Topo Ⅱ proteins,and clinical pathological features were analyzed.Results The expression levels of COX-2 and VEGF in TNBC were higher than those in non-TNBC (78.3％ vs.66.7％,and 87.0％ vs.84.5％),but there was no statistically sighificant difference (P ＞0.05).The expression of Topo Ⅱ in TNBC was 91.3％,higher than that in non-TNBC (71.4％) (P ＜0.05).The expression of VEGF in TNBC was correlated with lymph node metastasis (P ＜ 0.05),but COX-2 and Topo Ⅱ were not.The expression of COX-2,VEGF and Topo Ⅱ was not correlated with the age,the size of tumor,the type of pathology,histology grade and vascular invasion (P ＞ 0.05).The expression of COX-2,VEGF and Topo Ⅱ in TNBC was correlated each other.Conclusion In TNBC,Topo Ⅱ has a significantly up-regulated expression.The expression of VEGF was correlated with lymph node metastasis.There are correlations among COX-2,VEGF and Topo Ⅱ,which maybe play an important role in the occurrence and development of TNBC. Key words: Triple-negative breast cancer; Cyclooxygenase-2; Vascular endothelial growth factor; Topoisomerase Ⅱ

Expression of DNA topoisomerase II-α: Clinical significance in laryngeal carcinoma.

DNA topoisomerase II-α (Topo II-α) is essential for numerous cell processes, including DNA replication, transcription, recombination, and chromosome separation and condensation. Altered Topo II-α expression may lead to carcinogenesis and cancer progression. The aim of the present study was to investigate the association between Topo II-α expression levels and clinicopathological data from laryngeal cancer patients. Immunohistochemistry was used to analyze Topo II-α expression in laryngeal squamous cell carcinoma and distant healthy tissues obtained from 70 patients. In addition, fluorescence in situ hybridization was used to detect Topo II-α amplification and chromosome 17 ploidy using a laryngeal cancer tissue microarray. The expression of Topo II-α protein was detected in 71.43% (50/70) of laryngeal carcinoma tissues, in contrast to 9% of healthy tissues (2/22). Furthermore, the expression of Topo II-α protein was found to be associated with tumor de-differentiation and advanced tumor T stage. However, the expression of Topo II-α protein was not identified to be associated with Topo II-α amplification in laryngeal carcinoma, although was found to positively correlate with chromosome 17 aneuploidy (P<0.05). A higher aneuploidy rate contributed to increased expression levels of Topo II-α protein. Aberrant Topo II-α expression and chromosome 17 aneuploidy contributed to the development and progression of laryngeal cancer, indicating that targeting Topo II-α may provide a treatment strategy for patients with laryngeal cancer.

Clinicopathological characteristics and expression of Topo IIα, Ki67 in triple negative breast cancer

Objective To investigate the clinicopathological characteristics and the expression of Topo Ⅱ α,Ki67 in triple negative breast cancer (TNBC).Methods The clinicopathological characteristics in TNBC group were analyzed,including year,tumor size,lymph node metastasis and histological type; the expression of Topo Ⅱ α and Ki67 was determined by immunohistochemical techniques (EnVision).And we compared the differences between TNBC group (78 cases) and the non-triple negative breast cancer (nonTNBC) group (114 cases).Results The positive rate of axillary lymph node metastasis in TNBC group was higher than that in non-TNBC group,which difference was significant (P ＜ 0.05) ; With the increase of the tumor size,the positive rate of axillary lymph node metastasis in TNBC group was significantly higher,The positive rate of axillary lymph node metastasis was correlated positively with the tumor size (P ＜ 0.05) ;the expression of Ki67 in non-TNBC group was significantly lower than that in TNBC group (x 2=4.003,P=0.045).Conclusion Compared with non-TNBC group patients,TNBC group patients have a higher risk of local invasion,lymph node metastasis,cell proliferation and poorer survival. Key words: Breast neoplasm; Triple negative; Topo Ⅱα ; Ki67

Role of topoisomerase I and thymidylate synthase expression in sporadic colorectal cancer: Associations with clinicopathological and molecular features

Topoisomerase I (Topo I) and thymidylate synthase (TS) are essential enzymes for the replication, transcription and repair of DNA, and are potential biomarkers in colorectal cancer (CRC). The aim of the study was to correlate the tissue expression of Topo I and TS in sporadic CRCs with relevant pathological and molecular features and patients’ outcome.Topo I and TS expression was assessed by immunostaining in 112 consecutive primary CRCs. Increased expression of Topo I was found in 36% of tumors, preferentially rectal (50%) and with not otherwise specified (NOS) histology (44%). Topo I expression was associated with 18q allelic loss (LOH), (p=0.013), microsatellite stable phenotype (p=0.002) and normal expression of mismatch proteins hMLH1 and hMSH2 (p=0.0012 and p=0.02, respectively). High TS expression was found in 60% of tumors, more frequently in distal sites (62%) and with NOS histology (66%); no association with microsatellite instability was observed.Topo I seems to be involved in the chromosomal instability pathway of sporadic CRCs. Conversely, high TS expression is unlikely to affect the clinical behavior of microsatellite unstable CRCs.