Lookup NU author(s): Dr Kay Padget,
Professor Andrew Pearson,
Professor Caroline Austin
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DNA topoisomerase II (topo II) is an essential nuclear enzyme and is the target for etoposide, which is used in the therapy of childhood acute lymphoblastic leukaemia (ALL). Topo II exists as two isoforms referred to as topo II alpha and topo II beta. To determine whether cellular levels of topo II alpha and beta are an important factor in determining drug sensitivity/resistance requires accurate, precise measurements of the two isoforms. We have developed a quantitative Western blotting method to accurately measure the absolute amounts of human topo II alpha and beta, using recombinant human topo II alpha and beta as standards. This quantitative method has been used to assess the efficiency of several commonly used topo II extraction protocols. The extractable amount of topo II alpha and alpha was found to be salt-dependent. However extraction using the optimal salt concentration was found to be as efficient as extraction with DNase I/Rnase A digestion and SDS solubilisation. Using the optimum extraction procedure and the quantitative immunoblotting method, topo II alpha and beta was quantified in cell lines, peripheral blood lymphocytes and in lymphoblasts from children with newly diagnosed ALL.
Author(s): Padget K, Pearson ADJ, Austin CA
Publication type: Article
Publication status: Published
ISSN (print): 0887-6924
ISSN (electronic): 1476-5551
PubMed id: 11069037
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