Takatoshi Ohno

Global Gene Expression Profiling of Pleural Mesotheliomas: Overexpression of Aurora Kinases and P16/CDKN2A Deletion as Prognostic Factors and Critical Evaluation of Microarray-Based Prognostic Prediction

Most gene expression profiling studies of mesothelioma have been based on relatively small sample numbers, limiting their statistical power. We did Affymetrix U133A microarray analysis on 99 pleural mesotheliomas, in which multivariate analysis showed advanced-stage, sarcomatous histology and P16/CDKN2A homozygous deletion to be significant independent adverse prognostic factors. Comparison of the expression profiles of epithelioid versus sarcomatous mesotheliomas identified many genes significantly overexpressed among the former, including previously unrecognized ones, such as uroplakins and kallikrein 11, both confirmed by immunohistochemistry. Examination of the gene expression correlates of survival showed that more aggressive mesotheliomas expressed higher levels of Aurora kinases A and B and functionally related genes involved in mitosis and cell cycle control. Independent confirmation of the negative effect of Aurora kinase B was obtained by immunohistochemistry in a separate patient cohort. A role for Aurora kinases in the aggressive behavior of mesotheliomas is of potential clinical interest because of the recent development of small-molecule inhibitors. We then used our data to develop microarray-based predictors of 1 year survival; these achieved a maximal accuracy of 68% in cross-validation. However, this was inferior to prognostic prediction based on standard clinicopathologic variables and P16/CDNK2A status (accuracy, 73%), and adding the microarray model to the latter did not improve overall accuracy. Finally, we evaluated three recently published microarray-based outcome prediction models, but their accuracies ranged from 63% to 67%, consistently lower than reported. Gene expression profiling of mesotheliomas is an important discovery tool, but its power in clinical prognostication has been overestimated.

Development of porous apatite ceramic for local delivery of chemotherapeutic agents

An experimental study was conducted on a drug delivery system (DDS), using porous apatite ceramics (PAC): hydroxyapatite block (HAb) [Ca10(PO4)6(OH)2] having a porosity of 35-48% and pore size range of 50-300 microm, and beta-tricalcium phosphate block (TCP) [Ca3(PO4)2] having a porosity of 75-80% and pore size range of 100-400 microm, for sustained release of a chemotherapeutic agent. Methotrexate (MTX) was loaded in the pores of PAC blocks by centrifuging the blocks in MTX solution. Impregnation of MTX in PAC blocks (1 cm3) was confirmed by a magnetic resonance imaging (MRI) study using Gadolinium-DTPA enhancement. The MRI showed high signal intensity in the PAC, which was confirmed by dye loading into the pores. To estimate the MTX-releasing capability of the PAC, the blocks were stored in 3 mL of phosphate-buffered saline (PBS) at 37 degrees C and the PBS was replaced every 48 h. The amount of MTX released was assayed by high-performance liquid chromatography. This study showed that MTX-impregnated PAC (0.63-2.25 mg/block) released the drug in a steady manner and maintained its concentration (0.1-1.0 microg/mL) up to 12 days. This concentration is high enough to be effective against tumor cells. Chemotherapeutic agent-impregnated PAC, prepared by simple centrifugation, could be a valuable form of local chemotherapy when used as a strut graft to repair bone defects. This new DDS material could also be used as an adjuvant to extended curettage and provide a means to reduce the recurrence of tumors without risk of systemic toxicity.

Small Interfering RNAs Expressed from a Pol III Promoter Suppress the EWS/Fli-1 Transcript in an Ewing Sarcoma Cell Line

The EWS/Fli-1 fusion gene encodes an oncogenic fusion protein. The fusion is a product of the translocation t(11;22) (q24;q12), which is detected in 85% of Ewing sarcoma and primitive neuroectodermal tumor cells. Utilizing intracellularly expressed 21- to 23-nucleotide small interfering RNAs (siRNAs) targeting the EWS/Fli-1 fusion transcript in an Ewing sarcoma cell line, we achieved a greater than 80% reduction in the EWS/Fli-1 transcript. The reduction in transcript levels was accompanied by growth inhibition of an Ewing cell line. In addition to quantitating the reduction of the fusion transcript, we carefully monitored reduction of the endogenous EWS and Fli-1 mRNAs as well. One of the two siRNAs targeted to the fusion transcript also partially downregulated the Fli-1 mRNA, further potentiating the growth inhibition. These results highlight both the power of siRNAs and the potential side reactions that need to be carefully monitored. In addition, these results provide the first demonstration of expressed siRNAs downregulating an oncogenic fusion transcript. The results and observations from these studies should prove useful in targeting other fusion transcripts characteristic of sarcomas and erythroleukemias.

The dangers of snowboarding: a 9-year prospective comparison of snowboarding and skiing injuries.

We studied 2,552 snowboarding injuries and 5048 skiing injuries sustained during 1988-97. The number of snowboarding injuries had been increasing year by year and was 6 times as many as skiing injuries (2.0 versus 0.35 per 1,000 visits). The types of snowboarding injuries included fractures (39%), lacerations (21%), dislocations (17%), and contusions (15%). Upper extremity injuries were more frequent than those in the lower extremity in snowboarders. The commonest fractures involved the radius (48%), clavicle (11%), humerus (11%), and ulna (7-8%). The shoulder joint was most commonly dislocated (55%) followed by the elbow (27%), acromioclavicular (10%), finger (4%), and hip joints. In snowboarding accidents, the rates of fractures and dislocations were higher than those in skiing in almost every part of the body. Severe injuries were commoner in snowboarding accidents. We recommend the use of appropriate equipment and instructions for beginners to prevent such injuries.

Inhibition of Platelet-derived Growth Factor-induced Cell Growth Signaling by a Short Interfering RNA for EWS-Fli1 via Down-regulation of Phospholipase D2 in Ewing Sarcoma Cells

EWS-Fli1, a fusion gene resulting from a chromosomal translocation t(11;22, q24;q12) and found in Ewing sarcoma and primitive neuroectodermal tumors, encodes a transcriptional activator and promotes cellular transformation. However, the precise biological functions of its products remain unknown. To investigate the role of EWS-Fli1 in cell growth signaling, we transfected Ewing sarcoma TC-135 cells with short interfering RNAs for EWS-Fli1. EWS-Fli1 knockdown reduced cell growth and platelet-derived growth factor (PDGF)-BB-induced activation of the growth signaling enzymes. Interestingly, phospholipase D2 (but not the PDGF-BB receptor) showed marked down-regulation in the EWS-Fli1-knocked down TC-135 cells compared with the control cells. In Ewing sarcoma TC-135 cells, the PDGF-BB-induced phosphorylation of growth signaling involving extracellular signal-regulated kinase, Akt, p70S6K, and the expression of cyclin D3 were markedly inhibited by transfection with short interfering RNA phospholipase (PL)-D2. The PDGF-BB-induced activation of growth signaling was also suppressed by 1-butanol, which prevents the production of phosphatidic acid by phospholipase D (but not by t-butyl alcohol), thereby implicating PLD2 in PDGF-BB-mediated signaling in TC-135 cells. These results suggest that EWS-Fli1 may play a role in the regulation of tumor proliferation-signaling enzymes via PLD2 expression in Ewing sarcoma cells.

EWS-Fli1 Up-Regulates Expression of the Aurora A and Aurora B Kinases

EWS-Fli1, a fusion gene resulting from the chromosomal translocation t(11;22, q24;q12), encodes a transcriptional activator, promotes cellular transformation, and is often found in Ewing sarcoma and primitive neuroectodermal tumor. The Aurora A and Aurora B kinases belong to a highly conserved family of serine/threonine protein kinases, are tightly regulated during the cell cycle, and are overexpressed in many carcinomas. Because the relationship between the Aurora A and/or Aurora B genes and the EWS-Fli1 fusion gene is unknown, we investigated the regulatory mechanism(s) by which Aurora kinases are controlled. Knockdown of EWS-Fli1 by small interfering RNA reduced mRNA levels not only of EWS-Fli1 but also of Aurora A and Aurora B. Luciferase assay using Aurora A and Aurora B promoters showed up-regulated activities compared with those of an empty vector. Experiments with deletion and point mutants showed positive regulatory Ets-binding sites located −84 and −71 bp upstream of the transcription initiation sites in Aurora A and Aurora B, respectively. Moreover, chromatin immunoprecipitation assay revealed that EWS-Fli1 gene products interact with both the Aurora A and Aurora B promoters. These results strongly suggest that the mitotic kinases Aurora A and Aurora B are regulated by EWS-Fli1 fusion protein in Ewing sarcoma cells. (Mol Cancer Res 2008;6(12):1937–45)

Synthetic siRNA targeting the breakpoint of EWS/Fli-1 inhibits growth of Ewing sarcoma xenografts in a mouse model

The EWS/Fli‐1 fusion gene, a product of the translocation t(11;22, q24;q12), is detected in 85% of Ewing sarcomas and primitive neuroectodermal tumors. It is thought to be a transcriptional activator that plays a significant role in tumorigenesis. In this study, we developed a novel EWS/Fli‐1 blockade system using RNA interference and tested its application for inhibiting the proliferation of Ewing sarcoma cells in vitro and the treatment of mouse tumor xenografts in vivo. We designed and synthesized a small interfering RNA (siRNA) possessing an aromatic compound at the 3′‐end targeting the breakpoint of EWS/Fli‐1. As this sequence is present only in tumor cells, it is a potentially relevant target. We found that the siRNA targeting EWS/Fli‐1 significantly suppressed the expression of EWS/Fli‐1 protein sequence specifically and also reduced the expression of c‐Myc protein in Ewing sarcoma cells. We further demonstrated that inhibition of EWS/Fli‐1 expression efficiently inhibited the proliferation of the transfected cells but did not induce apoptotic cell death. In addition, the siRNA possessing the aromatic compound at the 3′‐end was more resistant to nucleolytic degradation than the unmodified siRNA. Administration of the siRNA with atelocollagen significantly inhibited the tumor growth of TC‐135, a Ewing sarcoma cell line, which had been subcutaneously xenografted into mice. Moreover, modification of the 3′‐end with an aromatic compound improved its efficiency in vivo. Our data suggest that specific downregulation of EWS/Fli‐1 by RNA interference is a possible approach for the treatment of Ewing sarcoma.

EWS/Fli‐1 chimeric fusion gene upregulates vascular endothelial growth factor‐A

Vascular endothelial growth factor (VEGF)‐A plays an important role in the pathological angiogenesis that occurs in soft‐tissue sarcoma and in about half of Ewings sarcoma cases, where it is highly overexpressed. EWS/Fli‐1 is considered to be a transcriptional activator and to play a significant role in tumorigenesis of Ewings sarcoma. However, the relationship between EWS/Fli‐1 and VEGF‐A is still unclear. The aim of this research is to investigate the relationship between EWS/Fli‐1 and VEGF‐A, and to determine whether small interfering RNA (siRNA)‐targeting of VEGF‐A can be developed as a novel treatment for Ewings sarcoma. Knockdown of EWS/Fli‐1 using siRNA on a Ewings sarcoma cell line (A673) suppressed VEGF‐A expression, and transfection of EWS/Fli‐1 into a human osteosarcoma cell line increased VEGF‐A expression. To inhibit VEGF‐A secretion from Ewings sarcoma, we developed a chemically synthesized siRNA that targets VEGF‐A. Transfection of the VEGF siRNA into the Ewings sarcoma cell line significantly suppressed VEGF‐A secretion by up to 98% in vitro, compared with a control. In vivo, we established Ewings sarcoma xenograft models and performed intratumoral injection of the siRNA mixed with atelocollagen. We observed that the inhibition of tumor growth occurs in a dose‐dependent manner. Histological examination revealed decreased microvessel density and morphological change around microvessels in the Ewings sarcoma xenografts treated with the siRNA. It is considered that a combination of chemically synthesized siRNA that targets VEGF‐A and atelocollagen might be a novel and effective option for treating Ewings sarcoma that secretes VEGF‐A.

Nucleolar organizer regions in rat urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine

SummaryThe number of nucleolar organizer regions (NORs) stained by the one-step silver colloid method was measured in preneoplastic and neoplastic bladder lesions induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in rats. Male ACI/N rats, 6 weeks of age, were given 0.05% BBN in drinking water for 5, 8, 12, 18 and 30 weeks to induce preneoplastic and neoplastic transitional cell lesions. The mean numbers of silverstained NORs (AgNORs) in such lesions were as follows: untreated transitional epithelium (n = 6), 1.26 ±0.09; transitional cell epithelium outside focal lesions (n= 10), 1.75 ±0.10; simple hyperplasia (n= 10), 2.01 ±0.15; papillary or nodular (PN) hyperplasia (n= 10), 2.15±0.19; transitional cell papilloma (n= 5), 2.37 ±0.12; transitional cell carcinoma (n= 5), 3.52 ±0.23. Thus, the mean number of AgNORs showed a step-wise increase from untreated and treated, histologically normal transitional epithelium through simple hyperplasia and PN hyperplasia to transitional cell papilloma and carcinoma. These results suggest that the mean number of AgNORs may reflect the proliferative nature of bladder lesions induced by BBN, as reported in preneoplastic and neoplastic lesions in other organs. PN hyperplasias were classified into two types based upon the mean number of AgNORs, indicating that they include reversible and irreversible changes in contrast with simple hyperplasia which is reversible change.

The loss of susceptibility to apoptosis in exudated tissue neutrophils is associated with their nuclear factor-κB activation

Tissue neutrophils, human salivary neutrophils donated from healthy subjects and synovial fluid neutrophils collected from patients with rheumatoid arthritis were compared with circulating blood neutrophils. Concomitant treatment of circulating blood neutrophils with tumor necrosis factor-alpha (TNF-alpha) and cycloheximide induced neutrophil apoptosis, whereas the same treatment failed to induce significant apoptosis in salivary and synovial fluid neutrophils. Caspase-3 activation by TNF-alpha was observed in these tissue neutrophils, although its activity was significantly weaker than that in circulating blood neutrophils. In circulating blood neutrophils, TNF-alpha induced activation of nuclear factor-kappa B (NF-kappa B), whereas, in tissue neutrophils, NF-kappa B had been already activated without any stimulation, and no further activation was induced by the treatment with TNF-alpha. Furthermore, while pretreatment of neutrophils with an NF-kappa B inhibitor produced typical apoptotic changes in circulating blood neutrophils, this inhibitor did not produce any morphological apoptotic changes induced by TNF-alpha in tissue neutrophils. These results indicate that neutrophils undergo marked functional changes such as altered sensitivity to apoptosis-inducing stimuli in association with their exudation from blood into tissue, and that NF-kappa B activation is involved in the acquisition of resistance to TNF-alpha-induced apoptosis.