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 IIα and topo IIβ. To determine whether cellular levels of topo IIα and β 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 IIα and β, using recombinant human topo IIα and β 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 IIα and β 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 IIα and β was quantified in cell lines, peripheral blood lymphocytes and in lymphoblasts from children with newly diagnosed ALL.
|Publication status||Published - 23 Oct 2000|