Topoisomerase IIa protein expression analysis in oral squamous cell carcinoma.

PURPOSE: Topoisomerases represent a super-family of nucleic enzymes involved in the DNA replication, transcription, recombination, and also chromosome topological formation. Topoisomerase II alpha (Topo IIa-gene location 17q21) is a critical gene associated with response to chemotherapeutic agents such as anthracyclines especially in breast adenocarcinoma. Our aim was to investigate the role of aberrant Topo IIa protein expression in oral squamous cell carcinoma (OSCC). METHODS: Fifty formalin-fixed, paraffin-embedded primary OSCCs tissue sections were used. Immunohistochemistry was performed using an anti- Topo IIa antibody. Digital image analysis was implemented for evaluating objectively the protein expression levels on the corresponding stained nuclei. RESULTS: Topo IIa protein overexpression (moderate to high immunostaining intensity values) was observed in 29/50 (58%) tissue cores, whereas low expression rates were detected in the remaining cases (21/50;42%). Topo IIa overall expression was strongly associated with the differentiation grade of the examined tumors (p=0.037) and also with human papillomavirus (HPV) positivity (p=0.029). No other statistical correlations were identified. CONCLUSIONS: Topo IIa overexpression is observed in significant subsets of OSCCs correlated with the grade of differentiation. Additionally, HPV persistent infection is associated with increased Topo IIa protein expression levels. Topo IIa expression analysis should be critical for identifying patients eligible for applying specific chemotherapeutic strategies based on anti-Topo IIa agents.

Inhibition of topoisomerase IIA (Top2α) induces telomeric DNA damage and T cell dysfunction during chronic viral infection.

T cells play a critical role in controlling viral infection; however, the mechanisms regulating their responses remain incompletely understood. Here, we investigated the role of topoisomerase IIA (Top2α, an enzyme that is essential in resolving entangled DNA strands during replication) in telomeric DNA damage and T cell dysfunction during viral infection. We demonstrated that T cells derived from patients with chronic viral (HBV, HCV, and HIV) infection had lower Top2α protein levels and enzymatic activity, along with an accumulation of the Top2α cleavage complex (Top2cc) in genomic DNA. In addition, T cells from virally infected subjects with lower Top2α levels were vulnerable to Top2α inhibitor-induced cell apoptosis, indicating an important role for Top2α in preventing DNA topological disruption and cell death. Using Top2α inhibitor (ICRF193 or Etoposide)-treated primary T cells as a model, we demonstrated that disrupting the DNA topology promoted DNA damage and T cell apoptosis via Top2cc accumulation that is associated with protein-DNA breaks (PDB) at genomic DNA. Disruption of the DNA topology was likely due to diminished expression of tyrosyl-DNA phosphodiesterase 2 (TDP2), which was inhibited in T cells in vitro by Top2α inhibitor and in vivo by chronic viral infection. These results suggest that immune-evasive viruses (HBV, HCV, and HIV) can disrupt T cell DNA topology as a mechanism of dysregulating host immunity and establishing chronic infection. Thus, restoring the DNA topologic machinery may serve as a novel strategy to protect T cells from unwanted DNA damage and to maintain immune competence.

Elevated TOP2A and UBE2C expressions correlate with poor prognosis in patients with surgically resected lung adenocarcinoma: a study based on immunohistochemical analysis and bioinformatics.

PURPOSE: Lung cancer has the highest morbidity and mortality among all cancer types. Reliable prognostic biomarkers are needed to identify high-risk patients apart from TNM system for precision medicine. The present study is designed to identify robust prognostic biomarkers in lung adenocarcinoma (LUAD) based on integration of multiple GEO datasets, The Cancer Genome Atlas (TCGA) database and Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. METHODS: Four LUAD GEO datasets (GSE10072, GSE2514, GSE43458, and GSE32863) and TCGA database were implemented to analyze the differently expressed genes (DEGs). Gene ontology, KEGG pathway, and protein-protein interaction network (PPI) were conducted based on the above DEGs. Hub genes were selected based on connectivity degree in the PPI network. Expression analysis and Kaplan-Meier survival analysis were conducted in CPTAC lung adenocarcinomas cohort. Kaplan-Meier survival analysis and Cox proportional hazards regression were performed on these hub genes using TCGA and our own cohort. RESULTS: A total of 430 shared genes in all five datasets were identified as DEGs. Based on their PPI network, nine hub genes were selected and all of them were significantly associated with overall survival using GEPIA analysis. Two hub genes, TOP2A and UBE2C, were further combined and showed poorer prognosis in both TCGA dataset and our validated cohort. Analysis in CPTAC revealed that TOP2A and UBE2C were significantly highly expressed in tumor sample. Multivariable analysis suggested TOP2A and UBE2C as independent prognostic factors in LUAD. CONCLUSION: Using data mining approach, we identified TOP2A and UBE2C as two robust prognostic factors in LUAD. We also demonstrated the TOP2A/UBE2C co-expression status in LUAD, and TOP2A/UBE2C co-expression correlated with poorer prognosis. More in-depth research is needed for transforming this result into clinical setting.

hsa-miR-9-3p and hsa-miR-9-5p as Post-Transcriptional Modulators of DNA Topoisomerase IIα in Human Leukemia K562 Cells with Acquired Resistance to Etoposide.

DNA topoisomerase IIα protein (TOP2α) 170 kDa (TOP2α/170) is an important target for anticancer agents whose efficacy is often attenuated by chemoresistance. Our laboratory has characterized acquired resistance to etoposide in human leukemia K562 cells. The clonal resistant subline K/VP.5 contains reduced TOP2α/170 mRNA and protein levels compared with parental K562 cells. The aim of this study was to determine whether microRNA (miRNA)-mediated mechanisms play a role in drug resistance via decreased expression of TOP2α/170. miRNA-sequencing revealed that human miR-9-3p and miR-9-5p were among the top six of those overexpressed in K/VP.5 compared with K562 cells; validation by quantitative polymerase chain reaction demonstrated overexpression of both miRNAs. miRNA recognition elements (MREs) for both miRNAs are present in the 3'-untranslated region (UTR) of TOP2α/170. Transfecting K562 cells with a reporter plasmid harboring the TOP2α/170 3'-UTR together with either miR-9-3p or miR-9-5p mimics resulted in a statistically significant decrease in luciferase expression. Mutating the miR-9-3p or miR-9-5p MREs prevented this decrease, demonstrating direct interaction between these miRNAs and TOP2α/170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2α/170 protein levels without a change in TOP2α/170 mRNA and resulted in attenuated etoposide-induced DNA damage (gain-of-miRNA-inhibitory function). Conversely, transfection of miR-9-3p or miR-9-5p inhibitors in K/VP.5 cells (overexpressed miR-9 and low TOP2α/170) led to increased TOP2α/170 protein expression without a change in TOP2α/170 mRNA levels and resulted in enhancement of etoposide-induced DNA damage (loss-of-miRNA-inhibitory function). Taken together, these results strongly suggest that these miRNAs play a role in and are potential targets for circumvention of acquired resistance to etoposide. SIGNIFICANCE STATEMENT: Results presented here indicate that miR-9-3p and miR-9-5p decrease DNA topoisomerase IIα protein 170 kDa expression levels in acquired resistance to etoposide. These findings contribute new information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. Furthermore, increased expression of miR-9-3p and miR-9-5p in chemoresistant cancer cells may support their validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy.

The Deubiquitinating Enzyme Inhibitor PR-619 is a Potent DNA Topoisomerase II Poison.

2,6-Diaminopyridine-3,5-bis(thiocyanate) (PR-619) is a broad-spectrum deubiquitinating enzyme (DUB) inhibitor that has been employed in cell-based studies as a tool to investigate the role of ubiquitination in various cellular processes. Here, we demonstrate that in addition to its action as a DUB inhibitor, PR-619 is a potent DNA topoisomerase II (TOP2) poison, inducing both DNA topoisomerase IIα (TOP2A) and DNA topoisomerase IIß (TOP2B) covalent DNA complexes with similar efficiency to the archetypal TOP2 poison etoposide. However, in contrast to etoposide, which induces TOP2-DNA complexes with a pan-nuclear distribution, PR-619 treatment results in nucleolar concentration of TOP2A and TOP2B. Notably, neither the induction of TOP2-DNA covalent complexes nor their nucleolar concentration are due to TOP2 hyperubiquitination since both occur even under conditions of depleted ubiquitin. Like etoposide, since PR-619 affected TOP2 enzyme activity in in vitro enzyme assays as well as in live cells, we conclude that PR-619 interacts directly with TOP2A and TOP2B. The concentration at which PR-619 exhibits robust cellular DUB inhibitor activity (5-20 µM) is similar to the lowest concentration at which TOP2 poison activity was detected (above 20 µM), which suggests that caution should be exercised when employing this DUB inhibitor in cell-based studies.

Diffusion Kurtosis Imaging Reflects Glial Fibrillary Acidic Protein (GFAP), Topo IIα, and O6-Methylguanine-DNA Methyltransferase (MGMT) Expression in Astrocytomas.

BACKGROUND Astrocytomas are the most common primary brain neoplasms. Biological indicators of astrocytomas can reflect its biological characteristics. The aim of this study was to assess the expression of the pathological glial fibrillary acidic protein (GFAP) Topo IIα and O6-methylguanine-DNA methyltransferase (MGMT) in astrocytomas using magnetic resonance (MR) diffusion kurtosis imaging (DKI) to evaluate the biological characteristics of astrocytomas. MATERIAL AND METHODS Sixty-six patients with pathologically proven astrocytomas were enrolled in this study. All patients underwent conventional MRI head scanning, DKI scanning, and enhanced scanning under the same conditions. Spearman's rank correlation analysis and Bonferroni correction were used to compare the values of DKI and the expression levels of GFAP, Topo IIα, and MGMT between the 2 groups. RESULTS Mean kurtosis (MK) values were negatively correlated with the expression of GFAP (r=-0.836; P=0.03). However, these were positively correlated with the expression of Topo IIα (r=0.896; P=0.01). Moreover, fractional anisotropy (FA) values were not correlated with the expression of GFAP (r=0.366; P=0.05), Topo IIα (r=-0.562; P=0.05), or MGMT (r=-0.153; P=0.10). CONCLUSIONS MK was significantly associated with the expression of GFAP and Topo IIα. To a certain extent, applying DKI may show the biological behavior of tumor cell differentiation, proliferation activity, invasion, and metastasis, and guide individual treatment.

Expression of M2 macrophage markers YKL-39 and CCL18 in breast cancer is associated with the effect of neoadjuvant chemotherapy.

PURPOSE: High activity of enzyme TOP2a in tumor cells is known to be associated with sensitivity to anthracycline chemotherapy, but 20% of such patients do not show clinical response. Tumor microenvironment, including tumor-associated macrophages (TAM), is an essential factor defining the efficiency of chemotherapy. In the present study, we analyzed the expression of M2 macrophage markers, YKL-39 and CCL18, in tumors of breast cancer patients received anthracycline-based NAC. METHODS: Patients were divided into two groups according to the level of doxorubicin sensitivity marker TOP2a: DOX-Sense and DOX-Res groups. Expression levels of TOR2a, CD68, YKL-39 and CCL18 genes were analyzed by qPCR, the amplification of TOR2a gene locus was assessed by the microarray assay. Clinical and pathological responses to neoadjuvant chemotherapy were assessed. RESULTS: We found that the average level of TOP2a expression in patients of DOX-Sense group was almost 10 times higher than in patients of DOX-Res group, and the expression of CD68 was 3 times higher in the DOX-Sense group compared to DOX-Res group. We demonstrated that expression levels of M2-derived cytokines but not the amount of TAM is indicative for clinical and pathological chemotherapy efficacy in breast cancer patients. Out of 8 patients from DOX-Sense group who did not respond to neoadjuvant chemotherapy (NAC), 7 patients had M2+ macrophage phenotype (YKL-39+CCL18- or YKL-39-CCL18+) and only one patient had M2- macrophage phenotype (YKL-39-CCL18-). In DOX-Res group, out of 14 patients who clinically responded to NAC 9 patients had M2- phenotype and only 5 patients had M2+ macrophage phenotype. Among pathological non-responders in DOX-Sense group, 19 (82%) patients had M2+ tumor phenotype and only 4 (18%) patients had M2- phenotype. In DOX-Res group, all 5 patients who pathologically responded to NAC had M2 phenotype (YKL-39-CCL18-). Unlike the clinical response to NAC, the differences in the frequency of M2+ and M2- phenotypes between pathologically responding and non-responding patients within DOX-Sense and DOX-Res groups were statistically significant. CONCLUSIONS: Thus, we showed that in patients with breast cancer who received anthracycline-containing NAC the absence of clinical response is associated with the presence of M2+ macrophage phenotype (YKL-39-CCL18 + or YKL-39 + CCL18-) based on TOP2a overexpression data.

Correlations of ICAM-1 gene polymorphisms with susceptibility and multidrug resistance in colorectal cancer in a Chinese population.

BACKGROUND: Colorectal cancer (CRC) is a malignant gastrointestinal tumor with a high mortality rate, including both colon and rectal cancer. In order to provide clinical guidance for the treatment of CRC, this study is conducted to investigate the correlations of intercellular adhesion molecule 1 (ICAM-1) gene polymorphisms with susceptibility and multidrug resistance (MDR) of colorectal cancer (CRC). METHODS: A total of 195 patients with CRC were selected as the observation group and 188 healthy people enrolled as the control group. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was used to test ICAM-1 A13848G and K469E polymorphisms. The expressions of MDR-associated protein topoisomerase II (Topo II) and P-glycoprotein (P-gp) in CRC tissues were detected by immunohistochemistry. The analysis on association of clinical indexes of CRC patients with ICAM-1 gene polymorphisms was performed. RESULTS: The frequencies of KK genotype and K allele of K469E in the observation group were significantly higher than that in the control group. KE + EE genotype and E allele might be protective factors for CRC. The distribution of genotypes, K469E KK and KE+EE, was highly correlated with histologic grade of tumor differentiation. Compared with adjacent normal tissues, positive rates of Topo II and P-gp expression were significantly increased in CRC tissues. Topo II expression in CRC patients was positively associated with lymph node metastasis and depth of tumor invasion, whereas P-gp expression was only associated with depth of tumor invasion. Higher positive rates of Topo II and P-gp expression were observed in ICAM-1 K469E KK genotype carriers, indicating that ICAM-1 K469E KK genotype might be related to MDR in CRC. CONCLUSION: These findings in the current study suggested that ICAM-1 K469E polymorphism is highly correlated with susceptibility and MDR in CRC.

A novel topoisomerase 2a inhibitor, cryptotanshinone, suppresses the growth of PC3 cells without apparent cytotoxicity.

DNA topoisomerase 2, which is ubiquitously expressed in eukaryotic cells, is an essential nuclear enzyme that promotes cell survival by regulating DNA topology and chromatid separation. This enzyme has been validated as a target for anticancer agent screening. It can be poisoned by common chemotherapeutics, such as etoposide and doxorubicin, which leads to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. However, recent studies have suggested that the topoisomerase 2a isozyme is predominantly responsible for the carcinogenic side effects associated with etoposide and doxorubicin chemotherapy. Thus, we need to find a promising topoisomerase 2-targeting anticancer agent that avoids these carcinogenic side effects. Recent studies have found that cryptotanshinone has obvious anticancer activities against diverse cancer cells. Here, we demonstrate that cryptotanshinone markedly decreases the steady-state mRNA level of topoisomerase 2a, thereby decreasing the protein and activity levels of this enzyme. Moreover, cryptotanshinone exhibited dramatic in vitro and in vivo antitumor activity with low toxicity to normal tissues. Collectively, our findings support the development of cryptotanshinone as a promising candidate for treating cancer by targeting topoisomerase 2a.

Immunohistochemical investigation of topoIIß, H3K27me3 and JMJD3 expressions in medulloblastoma.

Topoisomerase IIß (topoIIß) is a nuclear enzyme specifically expressed in neurons, and plays an important role in the development of the cerebellum. To date, the expression of topoIIß protein in medulloblastoma (MB) has not been investigated. In this study, 16 MB specimens including 10 classical subtypes of MB and 6 desmoplastic subtypes of MB (DMB), along with 5 normal cerebellum samples, were obtained from clinics. With immunohistochemical staining, prominently expressed topoIIß was seen in normal cerebellar tissues, while there was no or less pronounced staining in classical MB cells. Interestingly, on comparing topoIIß expression in different regions of DMB samples, relatively high levels of topoIIß were revealed within nodules composed of differentiated neurocytic cells, which are known to predict a favorable clinical outcome for MB. We also examined the expression of two epigenetic factors, H3K27me3 and JMJD3 in the different tissues. Very high levels of H3K27me3 were found in all MB samples, except the intranodules of DMB, where JMJD3 expression was more prominent. Furthermore, a negative correlation between topoIIß and H3K27me3 in MB was revealed in this study. Thus, our data primarily indicate that topoIIß can be used to estimate neuronal differentiation in MB, and may serve as a target for improving the survival rates for this condition. We speculate that H3K27me3 repression of topoIIß at the transcriptional level may occur, although this needs to be verified using larger numbers of MB samples in future experiments.

Topoisomerase 2α and thymidylate synthase expression in adrenocortical cancer.

Topoisomerase II alpha (TOP2A) and thymidylate synthase (TS) are known prognostic parameters in several tumors and also predictors of efficacy of anthracyclines, topoisomerase inhibitors and fluoropirimidines, respectively. Expression of TOP2A and TS mRNA was assessed in 98 patients with adrenocortical carcinoma (ACC) and protein expression was assessed by immunohistochemistry in a subset of 39 tumors. Ninety-two patients were radically resected for stage II-III disease and 38 of them received adjuvant mitotane. Twenty-six patients with metastatic disease received the EDP-M (etoposide, doxorubicin, Adriamycin, cisplatin plus mitotane). TOP2A and TS expression in ACC tissue was directly correlated with the clinical data. Both markers were not associated with either disease free survival (DFS) or overall survival (OS) in multivariate analyses and failed to be associated to mitotane efficacy. Disease response or stabilization to EDP-M treatment was observed in 12/17 (71%) and 1/9 (11%) patients with high and low TOP2A expressing tumors (P = 0.0039) and 9/13 (69%) and 4/13 (31%) patients with high and low TS expressing ACC, respectively (P = 0.049). High TOP2A expression was significantly associated with longer time to progression (TTP) after EDP-M. TOP2A and TS proteins assessed by immunohistochemistry significantly correlated with mRNA expression. Immunohistochemical TOP2A expression was associated with a non-significant better response and longer TTP after EDP-M. TOP2A and TS were neither prognostic nor predictive of mitotane efficacy in ACC patients. The predictive role of TOP2A expression of EDP-M activity suggests a significant contribution of Adriamycin and etoposide for the efficacy of the EDP scheme.

Prognostic value of survivin and DNA topoisomerase IIα in diffuse and anaplastic astrocytomas.

Distinguishing WHO grade II astrocytomas from grade III is a difficult task. This study looks into the potential prognostic use of mitotic activity and the proliferation markers Ki67/MiB-1 (Ki67), survivin and DNA topoisomerase IIα (TIIα) in 59 WHO grade II diffuse astrocytomas (DA) and 33 WHO grade III anaplastic astrocytomas (AA), IDH1 R132H-mutated and not otherwise specified (NOS) by means of immunohistochemistry. All proliferation markers showed higher expression in AA compared with DA. Only Ki67 had significantly greater expression in astrocytomas, NOS vs. astrocytomas, IDH1-mutated. Uni-/multivariable COX-regression analyses showed that greater expression of both survivin and TIIα were associated with poorer survival when stratified for IDH1-mutation status and, additionally, achieved hazard rates surpassing clinically established prognostic factors such as age and WHO performance status. Ki67 achieved only statistical significance in univariable analyses, whereas mitoses did not reveal any relation to survival. IDH1-mutated astrocytomas had significantly better survival than astrocytomas, NOS. Among IDH1-mutated astrocytomas no significant difference in survival was shown between DA and AA. Our findings suggest a potential usefulness of proliferation markers in the prognostic setting of astrocytomas independent of IDH1-mutation status, and survivin and TIIα are potential candidates in that regard.

Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide.

BACKGROUND: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. METHODS: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. RESULTS: Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2µM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. CONCLUSIONS: Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. GENERAL SIGNIFICANCE: Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Clinicopathological Significance of the Proliferation Markers Ki67, RacGAP1, and Topoisomerase 2 Alpha in Breast Cancer.

Objectives The aims of this study are to evaluate expressions of Ki67, RacGAP1 (MgcRacGAP) and topoisomerase 2 alpha (TOP2a), the markers related with cell proliferation that have been proposed to affect the prognosis in the literature and correlate the results with clinicopathological parameters of breast cancer patients. Methods Ki67, RacGAP1, and TOP2a antibodies were applied immunohistochemically to the tissue micrarray blocks of 457 female breast cancer patients. The results were correlated with clinical, prognostic, histopathological features, and other immunohistochemical findings (estrogen receptor [ER], progesterone receptor [PR], HER2, cytokeratin [CK]5/6, CK14, epidermal growth factor receptor [EGFR] and vimentin), statistically. Results Ki67 expression demonstrated direct correlation with TOP2a expression, mitotic count, tumor grade, geographic necrosis, basal-like phenotype. RacGAP1 expression was directly correlated with TOP2a expression, nipple invasion, and number of metastatic lymph nodes, and it was inversely correlated with PR expression. TOP2a expression was directly correlated with vimentin and Ki67 expressions, mitotic count, tumor grade, and geographic necrosis, and nipple invasion, and negatively correlated with ER and PR expressions. Higher TOP2a and Ki67 expressions were correlated with shorter overall survival. Higher TOP2a expression and RacGAP1 positivity were directly correlated with shorter disease-free survival. Conclusion This study showed that the overexpressions of Ki67, RacGAP1, and TOP2a affect the prognosis adversely, thus to develop target therapies against RacGAP1 and TOP2a as well as using Ki67 as a part of routine pathology practice might be beneficial in breast cancer therapy and prediction of prognosis.

Regulation of Poly(A) Tail and Translation during the Somatic Cell Cycle.

Poly(A) tails are critical for mRNA stability and translation. However, recent studies have challenged this view, showing that poly(A) tail length and translation efficiency are decoupled in non-embryonic cells. Using TAIL-seq and ribosome profiling, we investigate poly(A) tail dynamics and translational control in the somatic cell cycle. We find dramatic changes in poly(A) tail lengths of cell-cycle regulatory genes like CDK1, TOP2A, and FBXO5, explaining their translational repression in M phase. We also find that poly(A) tail length is coupled to translation when the poly(A) tail is <20 nucleotides. However, as most genes have >20 nucleotide poly(A) tails, their translation is regulated mainly via poly(A) tail length-independent mechanisms during the cell cycle. Specifically, we find that terminal oligopyrimidine (TOP) tract-containing transcripts escape global translational suppression in M phase and are actively translated. Our quantitative and comprehensive data provide a revised view of translational control in the somatic cell cycle.

Gastrointestinal stromal tumors - quantitative detection of the Ki-67, TPX2, TOP2A, and hTERT telomerase subunit mRNA levels to determine proliferation activity and a potential for aggressive biological behavior.

Gastrointestinal stromal tumors (GISTs) have an unpredictable biological potential ranging from benign to malignant. Molecular markers involved in the mechanisms of proliferation and cellular senescence may provide additional information about biological behavior of the tumor. The aim of the present study was to investigate Ki-67, TPX2, TOP2A and hTERT mRNA expression levels in specimens from patients with GISTs to define relationships between proliferation activity and biological potential and progression of the disease. We measured Ki-67, TPX2, TOP2A and hTERT mRNA levels using quantitative real-time reverse transcription PCR (RQ RT PCR). The highest Ki-67, TPX2, TOP2A and hTERT mRNA expression levels were found in the highly proliferative BLs (18 specimens), in comparison with GISTs (137 specimens) and LMSs (9 specimens). Patients with GISTs and adequate information about mitotic activity, tumor size and anatomical site (84 specimens) were divided into two groups - GISTs with benign (29 patients) and with malignant (55 patients) potential. We observed association between higher Ki-67, TPX2 and hTERT mRNA levels and the GISTs with malignant potential. Univariate analysis (57 patients with available follow-up information) of survival (Kaplan Meier curves method) revealed a correlation between higher levels of TPX2, Ki-67 and hTERT markers and shorter event-free survival (EFS) or poorer overall survival (OS). The results demonstrate the importance of quantitative assessment of the proliferation activity in GISTs. Proliferation markers of Ki-67, TPX2, TOP2A and hTERT are suitable markers for detection the proliferation activity and telomerase activity of these tumors. Furthermore, the assessment of TPX2, Ki-67 and hTERT expression levels is appropriate for determination of malignant potential of GISTs.

MRN, CtIP, and BRCA1 mediate repair of topoisomerase II-DNA adducts.

Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein-DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)-DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2-DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication.

Clinicopathological and prognostic significance of RECQL5 helicase expression in breast cancers.

RECQL5 is a member of the RecQ family of DNA helicases and has key roles in homologous recombination, base excision repair, replication and transcription. The clinicopathological significance of RECQL5 expression in breast cancer is unknown. In this study, we have evaluated RECQL5 mRNA expression in 1977 breast cancers, and RECQL5 protein level in 1902 breast cancers [Nottingham Tenovus series (n = 1650) and ER- cohort (n = 252)]. Expression levels were correlated to aggressive phenotypes and survival outcomes. High RECQL5 mRNA expression was significantly associated with high histological grade (P = 0.007), HER2 overexpression (P = 0.032), ER+/HER2-/high proliferation genefu subtype (P < 0.0001), integrative molecular clusters (intClust 1and 9) (P < 0.0001) and poor survival (P < 0.0001). In subgroup analysis, high RECQL5 mRNA level remains significantly associated with poor BCSS in ER+ cohort (P < 0.0001) but not in ER- cohort (P = 0.116). At the protein level, in tumours with low RAD51, high RECQL5 level was significantly associated with high histological grade (P < 0.0001), higher mitotic index (P = 0.008), dedifferentiation (P = 0.025), pleomorphism (P = 0.027) and poor survival (P = 0.003). In subgroup analysis, high RECQL5/low RAD51 remains significantly associated with poor BCSS in ER+ cohort (P = 0.010), but not in ER- cohort (P = 0.628). In multivariate analysis, high RECQL5 mRNA and high RECQL5/low RAD51 nuclear protein coexpression independently influenced survival (P = 0.022) in whole cohort and in the ER+ subgroup. Preclinically, we show that exogenous expression of RECQL5 in MCF10A cells can drive proliferation supporting an oncogenic function for RECQL5 in breast cancer. We conclude that RECQL5 is a promising biomarker in breast cancer.

Distinct expression patterns for type II topoisomerases IIA and IIB in the early foetal human telencephalon.

TOP2A and TOP2B are type II topoisomerase enzymes that have important but distinct roles in DNA replication and RNA transcription. Recently, TOP2B has been implicated in the transcription of long genes in particular that play crucial roles in neural development and are susceptible to mutations contributing to neurodevelopmental conditions such as autism and schizophrenia. This study maps their expression in the early foetal human telencephalon between 9 and 12 post-conceptional weeks. TOP2A immunoreactivity was restricted to cell nuclei of the proliferative layers of the cortex and ganglionic eminences (GE), including the ventricular zone and subventricular zone (SVZ) closely matching expression of the proliferation marker KI67. Comparison with sections immunolabelled for NKX2.1, a medial GE (MGE) marker, and PAX6, a cortical progenitor cell and lateral GE (LGE) marker, revealed that TOP2A-expressing cells were more abundant in MGE than the LGE. In the cortex, TOP2B is expressed in cell nuclei in both proliferative (SVZ) and post-mitotic compartments (intermediate zone and cortical plate) as revealed by comparison with immunostaining for PAX6 and the post-mitotic neuron marker TBR1. However, co-expression with KI67 was rare. In the GE, TOP2B was also expressed by proliferative and post-mitotic compartments. In situ hybridisation studies confirmed these patterns of expression, except that TOP2A mRNA is restricted to cells in the G2/M phase of division. Thus, during early development, TOP2A is likely to have a role in cell proliferation, whereas TOP2B is expressed in post-mitotic cells and may be important in controlling expression of long genes even at this early stage.

CD99 correlates with low cyclin D1, high topoisomerase 2 status and triple negative molecular phenotype but is prognostically irrelevant in breast carcinoma.

CD99 is a protein initially described in the Ewing sarcoma family of tumors, but growing evidence has shown its expression in other tumors of mesenchymal, hematopoietic and even epithelial origin. Some articles report CD99 in metaplastic carcinoma of the breast, a subtype of breast carcinoma (BC) with pronounced epithelial to mesenchymal (EMT) phenotype. Our aim was to analyse the potential relationship between CD99 and selected EMT (vimentin, E-cadherin, Twist) and proliferation markers (Ki-67, c-myc, cyclin D1, topoisomerase 2), molecular subtypes of BC, as well as overall survival (OS) and progression-free survival (PFS). In a group of 122 cases CD99 membrane expression was seen in 14 (11.5%) cases: strong in 11 (9%) and moderate in 3 (2.5%). Expression of CD99 correlated with low cyclin D1 index, high level of topoisomerase 2 expression and lack of progesterone receptor (PR) but not with EMT characteristics. Additionally, strong expression of CD99 correlated with triple negative molecular BC phenotype. CD99 was prognostically irrelevant for OS and PFS. CD99 correlates with selected proliferative markers and low ER/PR receptor status but not with patients' outcome in BC. Further studies are required to explain precisely its role in molecular pathogenesis of BC.