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Title: Regulation of RNA translation by phenethyl isothiocyanate
Author: Donlevy, Alison
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2013
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Phenethyl isothiocyanate (PEITC) is a dietary phytochemical that has received considerable interest for its potential chemopreventive/therapeutic anti -cancer activity. PEITC inhibits cancer cell proliferation and/or survival in vitro, suppresses angiogenesis and decreases tumour growth in vivo with little toxicity. However, the mechanisms by which PEITC exerts its anti-cancer effects are not known. The goal of this project was to investigate the hypothesis that anti-cancer effects ofPEITC may involve inhibition of mRNA translation. Effects of PEITC on global mRNA translation were first studied in human MCF7 breast cancer cells using both polysome proflling and 35S-metabolic labelling experiments. PEITC caused a dose- and time-dependent inhibition of mRNA translation, which was partially reversed following removal of PEITC. Inhibition of mRNA translation was associated with decreased expression of HIFla and VEGF, two proteins that are key for pro-angiogenic responses of malignant cells, in both normoxic and hypoxic conditions, at least in part via effects on translation of HlF1A and VEGF mRNAs. Although PEITC has previously been shown to inhibit signalling via the mTORC I pathway, further analysis demonstrated that PEITC also caused a rapid increase in phosphorylation of eIF2a at SerSI which can result in inhibition of initiation of mRNA translation. Increased eIF2a phosphorylation was important for PEITCmediated inhibition of mRNA translation since mouse embryo fIbroblasts expressing nonphosphorylatable eIF2a were relatively resistant to PEITC-induced inhibition of mRNA translation compared to control cells. In addition, PEITC caused the accumulation of stress granules, which have previously been associated with translationally stalled InRNAs. To extend these results to a more clinically relevant setting, further studies were performed using cells isolated fi'om the blood of patients with chronic lymphocytic leukaemia (CLL), the most common leukaemia in the Western world. Signalling via the B-cell receptor (BCR) is known to play a major role in the development and progression of CLL, and studies first investigated how BCR stimulation altered mRNA translation in primary CLL cells. Stimulation of surface IgM (sIgM) resulted in signiflcant, but variable, increases in mRNA translation. Overall, increases in InRNA translation were higher in samples that were considered as sIgM responsive (based on previous analysis of anti-IgM-induced intracellular Ca2+ mobilisation) compared to non-responsive samples, and in samples stimulated with anti-IgM compared to anti-IgD. Anti-IgM also increased expression of MYC and MCLl, two key targets for CLL proliferation and survival, via increased transcription and mRNA translation. ,PEITC inhibited both basal and anti-IgM-induced RNA translation, whereas ibrutinib and tamatinib, inhibitors of the BCR-associated signalling kinase BTK and SYK, predominantly inhibited antiIgM- induced mRNA translation. PEITC, ibrutinib and tamatinib also decreased translation of MYC and MCLl RNAs in anti-IgM treated cells. Similar to MCF7 cells, PEITC caused a rapid increase in eIF2a phosphorylation in CLL cells. Overall, these results are consistent with the hypothesis that inhibition of mRNA translation by PEITC may contribute to its anti-cancer effects. In particular, the work has revealed the eIF2a pathway as a novel target for PEITC and uncovered new links between translation and BCR signalling in human leukaemia. Inhibition of mRNA translation, in response to PEITC or novel kinase inhibitors may play an important role in the therapeutic effects of these agents.
Supervisor: Packham, Graham ; Coldwell, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR180 Immunology ; RC0254 Neoplasms. Tumors. Oncology (including Cancer)