http://bura.brunel.ac.uk/handle/2438/278 2013-05-21T21:21:18Z 2013-05-21T21:21:18Z Biggerstaff, John http://bura.brunel.ac.uk/handle/2438/6586 2012-09-21T12:08:24Z 2012-01-01T00:00:00Z

Title: The role of cytokines, coagulation and fibrinolysis in leucocyte and LAK cell cytotoxicity of tumour cells Authors: Biggerstaff, John Abstract: Interleukin-2 activates lymphocytes to become highly cytotoxic for a wide range of tumour cell types in vitro (Iymphokine activated killer or LAK cells), and in animal models. However, only limited therapeutic benefit was observed in clinical trials of LAK cell therapy. This project aimed to investigate the molecular and cellular interactions involved in the production and effector functions of LAK cells, to identify factor(s) which might be responsible for the poor clinical responses observed in LAK cell therapy. Tumour cell lines were heterogeneous in their response to killing by cytokines (TNFα, LT, IFNγ and IL-1β), and purified monocytes or lymphocytes, but were consistently highly sensitive to LAK cell cytotoxicity. Autologous monocytes and lymphocytes were not killed by LAK cells, in contrast to human umbilical vein endothelial cells and fibroblasts. Supernatants from LAK cells were considerably less cytotoxic than the effector cells, and physical separation of effector and target cells resulted in inhibition of killing. Lymphocyte and LAK cell cytotoxicity was associated predominantly with the CD8+ (cytotoxic T-cell) lymphocyte sub-population, and was significantly inhibited by anti-TNFα and anti-LT, demonstrating that these cytokines were the primary effector molecules in this system. LAK cells and A375 melanoma cells showed procoagulant activity, predominantly via the tissue factor pathway, and LAK cells also possessed surface factor V. In addition, A375 cells were highly fibrinolytic. Tumour cell killing by LAK cells was inhibited by plasma, and further experiments determined that polymerised fibrin, but not fibrin monomer was responsible. From these results it was suggested that culture of small numbers of cells from tumour biopsies, and the determination of their sensitivity to cytotoxic drugs, cytokines and effector cells may lead to more effective treatment protocols for immunotherapy of individual tumours. In order to enhance the efficacy of immunotherapy, further in vivo research is required to elucidate the interactions between immune effector cells and the coagulation/fibrinolytic systems. Description: This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.

2012-01-01T00:00:00Z Sottile, F Gnemmi, I Cantilena, S D'Acunto, WC Sala, A http://bura.brunel.ac.uk/handle/2438/6547 2013-02-08T12:58:37Z 2012-01-01T00:00:00Z

Title: A chemical screen identifies the chemotherapeutic drug topotecan as a specific inhibitor of the B-MYB/MYCN axis in neuroblastoma Authors: Sottile, F; Gnemmi, I; Cantilena, S; D'Acunto, WC; Sala, A Abstract: The transcription factor MycN is the prototypical neuroblastoma oncogene and a potential therapeutic target. However, its strong expression caused by gene amplification in about 30% of neuroblastoma patients is a considerable obstacle to the development of therapeutic approaches aiming at eliminating its tumourigenic activity. We have previously reported that B-Myb is essentially required for transcription of the MYCN amplicon and have also shown that B-MYB and MYCN are engaged in a feed forward loop promoting the survival/proliferation of neuroblastoma cells. We postulated that pharmacological strategies breaking the B-MYB/MYCN axis should result in clinically desirable effects. Thus, we implemented a high throughput chemical screen, using a curated library of ~1500 compounds from the National Cancer Institute, whose endpoint was the identification of small molecules that inhibited B-Myb. At the end of the screening, we found that the compounds pinafide, ellipticine and camptothecin inhibited B-Myb transcriptional activity in luciferase assays. One of the compounds, the topoisomerase-1 inhibitor camptothecin, is of considerable clinical interest since its derivatives topotecan and irinotecan are currently used as first and second line treatment agents for various types of cancer, including neuroblastoma. We found that neuroblastoma cells with amplification of MYCN are more sensitive than MYCN negative cells to camptothecin and topotecan killing. Campothecin and topotecan caused selective down-regulation of B-Myb and MycN expression in neuroblastoma cells. Notably, forced overexpression of B-Myb could antagonize the killing effect of topotecan and camptothecin, demonstrating that the transcription factor is a key target of the drugs. These results suggest that camptothecin and its analogues should be more effective in patients whose tumours feature amplification of MYCN and/or overexpression of B-MYB. Description: Copyright: © 2012 Sottile et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The article was made available through the Brunel Open Access Publishing Fund.

2012-01-01T00:00:00Z Cantilena, S Pastorino, F Pezzolo, A Chayka, O Pistoia, V Ponzoni, M Sala, A http://bura.brunel.ac.uk/handle/2438/6275 2013-02-08T12:38:43Z 2011-01-01T00:00:00Z

Title: Frizzled receptor 6 marks rare, highly tumourigenic stem-like cells in mouse and human neuroblastomas Authors: Cantilena, S; Pastorino, F; Pezzolo, A; Chayka, O; Pistoia, V; Ponzoni, M; Sala, A Abstract: Wnt signalling is an important component of vertebrate development, required for specification of the neural crest. Ten Wnt receptors [Frizzled receptor 1-10 (Fzd1-10)] have been identified so far, some of which are expressed in the developing nervous system and the neural crest. Here we show that expression of one such receptors, Fzd6, predicts poor survival in neuroblastoma patients and marks rare, HIF1/2 α-positive cells in tumour hypoxic areas. Fzd6 positive neuroblastoma cells form neurospheres with high efficiency, are resistant to doxorubicin killing and express high levels of mesenchymal markers such as Twist1 and Notch1. Expression of Fzd6 is required for the expression of genes of the noncanonical Wnt pathway and the spheres forming activity. When transplanted into immunodeficient mice, neuroblastoma cells expressing the Fzd6 marker grow more aggressively than their Fzd6 negative counterparts. We conclude that Fzd6 is a new surface marker of aggressive neuroblastoma cells with stem cell-like features. Description: Copyright © 2011 Cantilena et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The article was made available through the Brunel Open Access Publishing Fund.

2011-01-01T00:00:00Z Creton, S Aardema, MJ Carmichael, PL Harvey, JS Martin, FL Newbold, RF O'Donovan, MRO Pant, K Poth, A Sakai, A Sasaki, K Scott, AD Schechtman, LM Shen, RR Tanaka, N Yasaei, H http://bura.brunel.ac.uk/handle/2438/6112 2011-12-21T13:36:31Z 2012-01-01T00:00:00Z

Title: Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs Authors: Creton, S; Aardema, MJ; Carmichael, PL; Harvey, JS; Martin, FL; Newbold, RF; O'Donovan, MRO; Pant, K; Poth, A; Sakai, A; Sasaki, K; Scott, AD; Schechtman, LM; Shen, RR; Tanaka, N; Yasaei, H Abstract: Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting. Description: Copyright @ 2011 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2012-01-01T00:00:00Z