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A forward genetic screen to identify modifiers of chemotherapy using zebrafish- Study of rnaset2 deficiency in zebrafish
[Thesis]. Manchester, UK: The University of Manchester; 2010.
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Abstract
Chemotherapy frequently fails to cure cancer patients due to toxicity or resistance to treatment. Variability in toxicity and resistance is influenced by polymorphisms and mutations in the individual's genome (Pharmacogenetics). Zebrafish has extensive evolutionary conservation with human, its genetics is a powerful gene discovery tool, and it has been described as a suitable model in cancer research. To study chemotherapy resistance, we used ENU-mutagenised zebrafish in a forward genetic screen to identify genes that modify responses to cancer chemotherapy. Zebrafish larvae were challenged with two chemotherapeutic drugs and stained with acridine orange (AO) to detect apoptosis and reveal hypo- or hyper-responders to chemotherapy. A mutation, conferring an increased uptake of AO, was identified by genetic mapping as a premature stop codon truncating the ribonuclease T2 (rnaset2) gene. Human RNASET2 encodes a putative lysosomal RNase. Lysosomal storage disorders, due to deficiencies in lysosomal hydrolases and resultant accumulation of macromolecules within lysosomes, are collectively among the commonest genetic diseases. RNASET2 deficiency in man results in a static encephalopathy arising in infancy and characterized by multifocal bilateral white matter lesions, subcortical cysts and focal enlargement of the anterior inferior horn. This doctoral thesis demonstrates that rnaset2 deficient zebrafish embryos suffer from a lysosomal storage disorder accumulating undigested ribosomal RNA (rRNA) in enlarged lysosomes within neurons of the brain. Moreover, high-field intensity μMRI revealed white matter lesions in the brain of adult rnase2 mutant animals. Thus, this zebrafish mutant can be used as a model to study the abnormalities observed in RNASET2 deficient individuals. This model suggests that the leukoencephalopathy results from a lysosomal storage disorder and provides a preclinical model for further elaborating disease mechanisms and evaluating candidate therapeutics.RNASET2 has also been advanced as a candidate tumour suppressor in several solid tumours. Recombinant rnaset2 protein has been tested in the clinic as an anti-cancer cytotoxic agent, with anti-angiogenic properties. By combining the rnaset2 mutant presented here with a transgenic melanoma model developed in the laboratory, the tumour suppressor and angiogenic role for rnaset2 was refuted.